Live attenuated chimeric porcine circovirus vaccine

ABSTRACT

The present invention provides a novel chimeric porcine circovirus infectious DNA clone and live attenuated chimeric virus with the PCV2, preferably of subtype PCV2b, capsid gene integrated into a non-pathogenic PCV1 virus genome. In a particular embodiment, the PCV2 capids gene is of subtype PCV2b, the predominant subtype circulating in pigs worldwide. The attenuated chimeric virus, designated PCV1-2b, effectively protects pigs from PCV2b challenges, and can be used as a live vaccine, as well as an inactivated (killed) vaccine, that provides protection and cross protection against PCV2b and PCV2a subtypes infection. The live attenuated vaccine of the present invention is also effective protecting pigs from porcine circovirus-associated disease (PCVAD).

REFERENCE TO RELATED APPLICATION

This patent application claims the benefit of U.S. Provisional PatentApplication No. 61/314,362, filed on Mar. 16, 2010, whose disclosuresare hereby incorporated by reference in their entirety into the presentdisclosure.

FIELD OF INVENTION

The present invention relates to infectious DNA clone, live attenuatedand inactivated vaccines of porcine circovirus (PCV), particularlychimeric virus of PCV1 and PCV2, particularly subtype PCV2b, and methodsfor protecting against PCV infection and porcine circovirus-associateddisease (PCVAD).

BACKGROUND OF THE INVENTION

Porcine circovirus (PCV) is a small, non-enveloped DNA virus whichbelongs to the family Circoviridae (Todd, D. et al., 2005, Circoviridae,p. 327-334. In C. M. Fauquet et al (ed.), Virus Taxonomy: Eighth Reportof the International Committee on Taxonomy of Viruses, Elsevier AcademicPress, San Diego). Type 1 PCV (PCV1) was discovered as a contaminant ofthe porcine kidney PK-15 cell line in the mid-seventies (Tischer, I. etal., 1974, Characterization of papovavirus- and picornavirus-likeparticles in permanent pig kidney cell lines, Zentralbl Bakteriol Orig A226:153-67).

PCV1 was considered to be a non-pathogenic virus because inoculation ofpigs with the PK-15 cell line-derived PCV1 virus did not cause anydisease in pigs (Tischer, I. et al., 1986, Studies on epidemiology andpathogenicity of porcine circovirus, Arch Viroi 91:271-6). In 1997, avariant strain of PCV, designated PCV type-2 (PCV2), was discovered inpiglets with wasting disease in Canada (Allan, G. M. et al., 1998,Isolation of porcine circovirus-like viruses from pigs with a wastingdisease in the USA and Europe. J Vet Diagn Invest 10:3-10; Clark, E. G.,1997, Presented at the 28th Annual Meeting of the American Associationof Swine Practitioners; Ellis, J. et al., 1998, Isolation of circovirusfrom lesions of pigs with postweaning muitisystemic wasting syndrome,Can Vet J 39:44-51; Meehan, B. M. et al., 1998, Characterization ofnovel circovirus DNAs associated with wasting syndromes in pigs, J GenVirol 79 (Pt 9):2171-9). Currently, PCV2 is the primary causative agentof porcine circovirus-associated disease (PCVAD), which includeswasting, mortality, respiratory signs, enteritis, reproductive failure,and porcine dermatitis and nephropathy syndrome (PDNS) (Opriessnig, T.et al., 2007, Porcine circovirus type 2 associated disease: update oncurrent terminology, clinical manifestations, pathogenesis, diagnosis,and intervention strategies, J Vet Diagn Invest 19:591-615). PCV2 iscurrently considered to be one of the most economically-important viralpathogens in global pig populations, and is found in every major swineproducing country of the world (Gillespie, J. et al, 2009, PorcineCircovirus Type 2 and Porcine Circovirus-Associated Disease, J VetIntern Med). Observation of severe clinical PCVAD in conventional pigsexperimentally infected with PCV2 alone is uncommon, and coinfectionwith other swine pathogens such as porcine reproductive and respiratorysyndrome virus (PRRSV) or porcine parvovirus (PPV) is usually requiredto induce the full-spectrum of clinical PCVAD (Allan, G. M. et al.,2000, Experimental infection of colostrum deprived piglets with porcinecircovirus 2 (PCV2) and porcine reproductive and respiratory syndromevirus (PRRSV) potentiates PCV2 replication, Arch Virol 145:2421-9;Opriessnig, T. et al. 2007. Supra; Roca, M. et al., 2004, In vitro andin vivo characterization of an infectious clone of a European strain ofporcine circovirus type 2, J Gen Virol 85:1259-66; Rovira, A. et al.,2002, Experimental inoculation of conventional pigs with porcinereproductive and respiratory syndrome virus and porcine circovirus 2, JVirol 76:3232-9; Tomas, A. et al., 2008, A meta-analysis on experimentalinfections with porcine circovirus type 2 (PCV2), Vet Microbiol132:260-73). However, infection of caesarean-derived, colostrum-deprived(CD/CD) pigs with PCV2 alone has resulted in severe clinical PCVAD andmortality (Allan, G. et al. 2003, Reproduction of postweaningmuitisystemic wasting syndrome in pigs experimentally inoculated with aSwedish porcine circovirus 2 isolate, J Vet Diagn Invest 15:553-60;Allan, G. M. et al., 2004, PMWS: experimental model and co-infections,Vet Microbiol 98:165-8; Bolin, S. R. et al., 2001, Postweaningmultisystemic wasting syndrome induced after experimental inoculation ofcesarean-derived, colostrum-deprived piglets with type 2 porcinecircovirus, J Vet Diagn Invest 13:185-94; Harms, P. A. et al., 2001,Experimental reproduction of severe disease in CD/CD pigs concurrentlyinfected with type 2 porcine circovirus and porcine reproductive andrespiratory syndrome virus, Vet Pathol 38:528-39; Kennedy, S. et al.,2000, Reproduction of lesions of postweaning muitisystemic wastingsyndrome by infection of conventional pigs with porcine circovirus type2 alone or in combination with porcine parvovirus, J Comp Pathol122:9-24). Several comprehensive reviews of the pathogenesis,immunology, and molecular biology of PCV2 are available (Allan, G. M.and J. A. Ellis, 2000, Porcine circoviruses: a review. J Vet DiagnInvest 12:3-14; Ellis, J. et al., 2004, Porcine circovirus-2 andconcurrent infections in the field, Vet Microbiol 98:159-63;Finsterbusch, 1′. and A. Mankertz, 2009, Porcine circoviruses-small butpowerful, Virus Res 143:177-83; Gillespie, J. et al., 2009, supra;Mankertz, A. et al., 2004, Molecular biology of Porcine circovirus:analyses of gene expression and viral replication, Vet Microbiol98:81-8; Opriessnig, T. et al. 2007, Supra; Ramamoorthy, S, and X. J.Meng, 2009, Porcine circoviruses: a minuscule yet mammoth paradox, AnimHealth Res Rev 10:1-20; Segales, J. et al., 2005, Porcine circovirusdiseases, Anim Health Res Rev 6:119-42).

Although the genomic organization of the pathogenic PCV2 and thenon-pathogenic PCV1 is similar, the genomes of PCV1 and PCV2 share onlyapproximately 68-76% nucleotide sequence identity (Fenaux, M. et al.,2004, Detection and in vitro and in vivo characterization of porcinecircovirus DNA from a porcine-derived commercial pepsin product, J GenVirol 85:3377-82; Hamel, A. L et al., 1998, Nucleotide sequence ofporcine circovirus associated with postweaning muitisystemic wastingsyndrome in pigs, J Virol 72:5262-7; Tischer, I. et al., 1982, A verysmall porcine virus with circular single-stranded DNA, Nature 295:64-6)and differences in transcriptional patterns and antigenic profile of thecapsid protein have been reported (Cheung, A. K. 2003, Comparativeanalysis of the transcriptional patterns of pathogenic and nonpathogenicporcine circoviruses, Virology 310:41-9; Lekcharoensuk, P. et al., 2004,Epitope mapping of the major capsid protein of type 2 porcine circovirus(PCV2) by using chimeric PCV1 and PCV2, J Virol 78:8135-45; Shang, S. B.et al., 2009, Fine mapping of antigenic epitopes on capsid proteins ofporcine circovirus, and antigenic phenotype of porcine circovirus type2, Mol Immunol 46:327-34). The two major genes encoded by the viralgenome include the 942 bp replicase (rep) gene (Mankertz, A. and B.Hillenbrand, 2001, Replication of porcine circovirus type 1 requires twoproteins encoded by the viral rep gene, Virology 279:429-38) and the 702bp capsid gene (cap) (Nawagitgul, P. et al., 2000, Open reading frame 2of porcine circovirus type 2 encodes a major capsid protein, J Gen Virol81:2281-7). The rep gene is highly conserved between PCV1 and PCV2 withabout 83% nucleotide sequence identity while the cap gene shares onlyabout 67-70% identity (Mankertz, A. et al., 2004, supra). Currently, atleast three subtypes of PCV2 have been identified in swine herdsworldwide: PCV2a, PCV2b, and PCV2c (Dupont, K. et al., 2008, Genomicanalysis of PCV2 isolates from Danish archives and a current PMWScase-control study supports a shift in genotypes with time, VetMicrobiol 128:56-64; Segales, J. et al., 2008, PCV-2 genotype definitionand nomenclature, Vet Rec 162:867-8). PCV2a and PCV2b have both beenassociated with clinical PCVAD of varying degrees of severity (An, D. J.et al., 2007, Phylogenetic characterization of porcine circovirus type 2in PMWS and PDNS Korean pigs between 1999 and 2006, Virus Res129:115-22; Clacci-Zanella, J. R. et al, 2009, Detection of porcineCircovirus type 2 (PCV2) variants PCV2-1 and PCV2-2 in Brazilian pigpopulation, Res Vet Sci 87:157-60; Lager, K. M et al., 2007, Mortalityin pigs given porcine circovirus type 2 subgroup 1 and 2 viruses derivedfrom DNA clones, Vet Rec 161:428-9; Madson, D. M. et al., 2008,Characterization of shedding patterns of Porcine circovirus types 2a and2b in experimentally inoculated mature boars, J Vet Diagn invest20:725-34; Opriessnig, T. et al., 2006, Genetic and experimentalcomparison of porcine circovirus type 2 (PCV2) isolates from cases withand without PCV2-associated lesions provides evidence for differences invirulence, J Gen Virol 87:2923-32; Opriessnig, T. et al., 2008,Differences in virulence among porcine circovirus type 2 isolates areunrelated to cluster type 2a or 2b and prior infection providesheterologous protection, J Gen Virol 89:2482-91). Prior to 2005, onlyPCV2a was found within pig populations in the United States and Canada,while both PCV2a and PCV2b were present in Europe and China (Chae, J. S,and K. S. Choi, 2009, Genetic diversity of porcine circovirus type 2from pigs in Republic of Korea, Res Vet Sci; Dupont, K. et al., 2008,supra). Since 2005, novel PCV2b strains were recognized in the UnitedStates and there has been a global shift in a dominant prevalence ofPCV2b in pig populations, concurrently with increased severity ofclinical PCVAD (Carman, S. et al., 2008, The emergence of a new strainof porcine circovirus-2 in Ontario and Quebec swine and its associationwith severe porcine circovirus associated disease-2004-2006, Can J VetRes 72:259-68; Chae, J. S, and K. S. Choi, 2009, supra; Cheung, A. K. etal., 2007, Detection of two porcine circovirus type 2 genotypic groupsin United States swine herds, Arch Virol 152:1035-44; Clacci-Zanella, J.R. et al., 2009, supra; Dupont, K. et al., 2008, supra; Gagnon, C. A. etal., 2007, The emergence of porcine circovirus 2b genotype (PCV-2b) inswine in Canada, Can Vet J 48:811-9; Lipej, Z. et al., 2005, Postweaningmuitisystemic wasting syndrome (PMWS) in pigs in Croatia: detection andcharacterisation of porcine circovirus type 2 (PCV2), Acta Vet flung53:385-96; Wang, F. et al., 2009, Genetic variation analysis of Chinesestrains of porcine circovirus type 2, Virus Res 145:151-6; Wiederkehr,D. D. et al., 2009, A new emerging genotype subgroup within PCV-2bdominates the PMWS epizooty in Switzerland, Vet Microbiol 136:27-35).The pathogenicity of PCV2c is unclear, as it has only been reported innon-diseased herds in Denmark in 1980, 1987, and 1990 (Dupont, K. etal., 2008, supra).

The current available commercial vaccines are all killed or recombinantvaccines based upon the PCV2a subtype (Opriessnig, T. et al. 2007,Supra; Ramamoorthy, S, and X. J. Meng, 2009, supra). The inventors havepreviously successfully developed an inactivated vaccine, Suvaxyn PCV2®One Dose™, based upon the PCV1-2a chimeric virus (with the capsid geneof PCV2a in the backbone of PCV1) (Fenaux, M. et al., 2004A, A chimericporcine circovirus (PCV) with the immunogenic capsid gene of thepathogenic PCV type 2 (PCV2) cloned into the genomic backbone of thenonpathogenic PCV1 induces protective immunity against PCV2 infection inpigs, J Virol 78:6297-303; Fenaux, M. et al., 2003, Immunogenicity andpathogenicity of chimeric infectious DNA clones of pathogenic porcinecircovirus type 2 (PCV2) and nonpathogenic PCV1 in weanling pigs, JVirol 77:11232-43; Gillespie, J. et al., 2008, A genetically engineeredchimeric vaccine against porcine circovirus type 2 (PCV2) is geneticallystable in vitro and in vivo, Vaccine 26:4231-6). However, since thePCV2b subtype has now become the globally dominant genotype associatedwith severe clinical PCVAD in commercial pigs, and since PCV2a and PCV2bdiffer by as much as 10% nucleotide sequence identity (Fenaux, M. etal., 2000, Genetic characterization of type 2 porcine circovirus (PCV-2)from pigs with postweaning muitisystemic wasting syndrome in differentgeographic regions of North America and development of a differentialPCR-restriction fragment length polymorphism assay to detect anddifferentiate between infections with PCV-1 and PCV-2, J Clin Microbiol38:2494-503; Olvera, A. et al., 2007, Molecular evolution of porcinecircovirus type 2 genomes: phylogeny and clonality, Virology357:175-85), it is unknown whether the current PCV2a subtype-basedkilled or recombinant vaccines provide complete protection against thenewly-recognized PCV2b subtype. Several studies have demonstratedeffectiveness of current commercial vaccines against PCV2b challenge(Fort, M. et al., 2008, Porcine circovirus type 2 (PCV2) vaccination ofconventional pigs prevents viremia against PCV2 isolates of differentgenotypes and geographic origins, Vaccine 26:1063-71; Fort, M. et al.,2009, One dose of a porcine circovirus 2 (PCV2) sub-unit vaccineadministered to 3-week-old conventional piglets elicits cell-mediatedimmunity and significantly reduces PCV2 viremia in an experimentalmodel, Vaccine 27:4031-7; Opriessnig, T. et al., 2009, Comparison ofefficacy of commercial one dose and two dose PCV2 vaccines using a mixedPRRSV-PCV2-SIV clinical infection model 2-3-months post vaccination,Vaccine 27:1002-7), however it is imperative to develop a PCV2bsubtype-based vaccine, preferably a live-attenuated vaccine, againstPCVAD. A live-attenuated vaccine based on the new PCV2b subtype wouldpossibly provide much greater protection in the field than the currentavailable killed and subunit vaccines based on the PCV2a subtype.

SUMMARY OF THE INVENTION

The present invention provides a nucleic acid molecule of porcinecircovirus (PCV) comprising a nucleic acid molecule encoding anonpathogenic chimeric PCV derived from a genomic sequence of Type 1 PCV(PCV1), and at least a portion of an encoding sequence of a capsidprotein of Type 2 PCV (PCV2), preferably of subtype PCV2b.

In one embodiment of the present invention, the encoding sequence of acapsid protein of PCV2 is selected from the group consisting subtypesPCV2a, PCV2b, and PCV2c.

In another embodiment of the present invention, the encoding sequence ofa capsid protein of PCV2 is of subtype PCV2b.

In a further embodiment of the present invention, the encoding sequenceof a capsid protein of PCV2 is at least a portion of open reading frame2 (ORF2) of PCV2, preferably of subtype PCV2b.

In yet another embodiment of the present invention, the nucleic acidmolecule encoding more than one copy of a nonpathogenic chimeric PCVderived from a genomic sequence of Type 1 PCV (PCV1), and at least aportion of an encoding sequence of a capsid protein of Type 2 PCV(PCV2), preferably of subtype PCV2b.

The present invention also provides a biologically functional plasmid orviral vector containing a nucleic acid molecule encoding a nonpathogenicchimeric PCV derived from a genomic sequence of Type 1 PCV (PCV1), andat least a portion of an encoding sequence of a capsid protein of Type 2PCV (PCV2), preferably of subtype PCV2b.

The present invention further provides a suitable host cell transfectedby a vector comprising a nucleic acid molecule encoding a nonpathogenicchimeric PCV derived from a genomic sequence of Type 1 PCV (PCV 1), andat least a portion of an encoding sequence of a capsid protein of Type 2PCV (PCV2), preferably of subtype PCV2b.

The present invention further provides an avirulent, infectious chimericPCV produced by a suitable host cell transfected by a vector comprisinga nucleic acid molecule encoding a nonpathogenic chimeric PCV derivedfrom a genomic sequence of Type 1 PCV (PCV1), and at least a portion ofan encoding sequence of a capsid protein of Type 2 PCV (PCV2),preferably of subtype PCV2b.

The present invention further provides an inactivated chimeric PCVcomprising at least a portion of a capsid protein of Type 2 PCV (PCV2),preferably of subtype PCV2b.

In one embodiment of the present invention, the encoding sequence of acapsid protein of PCV2 is selected from the group consisting subtypesPCV2a, PCV2b, and PCV2c.

In another embodiment of the present invention, the encoding sequence ofa capsid protein of PCV2 is of subtype PCV2b.

In a further embodiment of the present invention, the encoding sequenceof a capsid protein of PCV2 is at least a portion of open reading frame2 (ORF2) of PCV2.

The present invention further provides a viral vaccine comprising aphysiologically acceptable carrier and an immunogenic amount of a memberselected from the group consisting of: (a) a nucleic acid molecule ofPorcine Circovirus (PCV) comprising a nucleic acid molecule encoding achimeric, nonpathogenic PCV derived from a genomic sequence of Type 1PCV (PCV1), and at least a portion of an encoding sequence of a capsidprotein of Type 2 PCV (PCV2), (b) a biologically functional plasmid orviral vector containing a nucleic acid molecule of PCV comprising anucleic acid molecule encoding a chimeric, nonpathogenic PCV derivedfrom a genomic sequence of PCV1, and at least a portion of an encodingsequence of a capsid protein of PCV2, (c) an avirulent, infectiousnonpathogenic chimeric PCV which contains a nucleic acid molecule of PCVcomprising a nucleic acid molecule encoding a chimeric, nonpathogenicPCV derived from a genomic sequence of PCV1, and at least a portion ofan encoding sequence of a capsid protein of PCV2, and (d) an inactivatedchimeric PCV comprising at least a portion of a capsid protein of PCV2,preferably of PCV2b subtype.

In one embodiment of the present invention, the vaccine contains livechimeric PCV virus.

In one embodiment of the present invention, the vaccine containsinactivated chimeric PCV virus.

In another embodiment of the present invention, the vaccine furthercontains an adjuvant.

In a further embodiment of the present invention, the vaccine protectsagainst PCV2a and PCV2b infection.

The present invention also provides a method of immunizing a pig againstPCV2 viral infection, comprising administering to a pig animmunologically effective amount of a viral vaccine comprising aphysiologically acceptable carrier and an immunogenic amount of a memberselected from the group consisting of: (a) a nucleic acid molecule ofPorcine Circovirus (PCV) comprising a nucleic acid molecule encoding achimeric, nonpathogenic PCV derived from a genomic sequence of Type 1PCV (PCV1), and at least a portion of an encoding sequence of a capsidprotein of Type 2 PCV (PCV2), (b) a biologically functional plasmid orviral vector containing a nucleic acid molecule of PCV comprising anucleic acid molecule encoding a chimeric, nonpathogenic PCV derivedfrom a genomic sequence of PCV1, and at least a portion of an encodingsequence of a capsid protein of PCV2, (c) an avirulent, infectiousnonpathogenic chimeric PCV which contains a nucleic acid molecule of PCVcomprising a nucleic acid molecule encoding a chimeric, nonpathogenicPCV derived from a genomic sequence of PCV 1, and at least a portion ofan encoding sequence of a capsid protein of PCV2, and (d) an inactivatedchimeric PCV comprising at least a portion of a capsid protein of PCV2,preferably of PCV2b subtype.

In one embodiment of the present invention, the method comprisingadministering the nucleic acid molecule or live attenuated chimeric PCVvirus to the pig.

In one embodiment of the present invention, the method comprisingadministering the inactivated chimeric PCV virus to the pig.

In another embodiment of the present invention, the method comprisingadministering the vaccine parenterally, intranasally, intradermally, ortransdermally to the pig.

In a further embodiment of the present invention, the method comprisingadministering the vaccine intralymphoidly or intramuscularly to the pig.

The present invention also provides a method of protecting a pig againstporcine circovirus-associated disease (PCVAD), comprising administeringto a pig an immunologically effective amount of a viral vaccinecomprising a physiologically acceptable carrier and an immunogenicamount of a member selected from the group consisting of: (a) a nucleicacid molecule of Porcine Circovirus (PCV) comprising a nucleic acidmolecule encoding a chimeric, nonpathogenic PCV derived from a genomicsequence of Type 1 PCV (PCV 1), and at least a portion of an encodingsequence of a capsid protein of Type 2 PCV (PCV2), (b) a biologicallyfunctional plasmid or viral vector containing a nucleic acid molecule ofPCV comprising a nucleic acid molecule encoding a chimeric,nonpathogenic PCV derived from a genomic sequence of PCV1, and at leasta portion of an encoding sequence of a capsid protein of PCV2, (c) anavirulent, infectious nonpathogenic chimeric PCV which contains anucleic acid molecule of PCV comprising a nucleic acid molecule encodinga chimeric, nonpathogenic PCV derived from a genomic sequence of PCV1,and at least a portion of an encoding sequence of a capsid protein ofPCV2, and (d) an inactivated chimeric PCV comprising at least a portionof a capsid protein of PCV2, preferably of subtype PCV2b.

BRIEF DESCRIPTION OF THE DRAWINGS

The above-mentioned features of the invention will become more clearlyunderstood from the following detailed description of the invention readtogether with the drawings in which:

FIG. 1 illustrates the construction and genomic organization offull-length PCV2b and chimeric PCV1-2b DNA clones. FIG. 1(A) shows aPCV2b monomeric DNA clone. Complete genome of wildtype PCV2b was clonedin pBSK+vector using unique Sac II site after amplification with PCRprimers A (SEQ ID No:3) and B (SEQ ID No:4)(Table 1). FIG. 1(B) showsChimeric PCV1-2b monomeric DNA clone. Chimeric PCV1-2b DNA clone wasconstructed by overlap extension PCR using PCR primers C—H (SEQ IDNo:5-10) (Table 1).

FIG. 2 illustrates PCV2 capsid-specific antibody response incaesarean-derived colostrum-deprived (CD/CD) pigs experimentallyinoculated with the chimeric PCV1-2b virus and the wildtype PCV2b virus.The mean serum S/P ratio+/−SEM is plotted for each treatment groupthroughout the experiment, with (*) indicating significant differenceson that day. Due to death or early euthanasia of some PCV2b-infectedpigs, fewer than five pigs were sampled in that group at 35-42 dpi.Groups with different letters are significantly different on that day.

FIG. 3 shows a comparison of the overall lymphoid lesion scores at 21dpi in the caesarean-derived colostrum-deprived (CD/CD) pigs inoculatedwith PBS buffer, chimeric PCV1-2b virus, and wildtype PCV2b virus.Combined lymphoid depletion, histiocytic replacement, and PCV2-specificantigen scores for the lymph nodes, spleen, and tonsil were comparedbetween treatments. The PCV2b-inoculated pig that died at 18 dpi wasincluded in the analysis. Circles indicate median, boxes are 1^(st) and3^(rd) quartiles, and whiskers indicate total range of data. Treatmentswith different letters are significantly different.

FIG. 4 illustrates the quantification of viral DNA loads in serum andlymphoid tissues in caesarean-derived colostrum-deprived (CD/CD) pigsexperimentally inoculated with the chimeric PCV1-2b virus and PCV2bvirus. FIG. 4(A) shows a quantification of viremia and viral DNA loadsin sera using qPCR. Group mean log viral genomic copies/ml ofserum+/−SEM is plotted for each treatment group, with (*) indicatingsignificant differences on that day. All samples at 0 dpi as well as allsamples from PBS-inoculated pigs were negative. Due to death or earlyeuthanasia of some PCV2b-infected pigs, fewer than five pigs weresampled in that group at 35-42 dpi. FIG. 4(B) shows a quantification ofviral DNA load in TBLN tissues using qPCR. Lymph node tissue viral DNAloads determined for each pig are plotted on the day they died or wereeuthanized, with group means+/−SEM plotted at 21 and 42 dpi. All samplesfrom PBS-inoculated pigs were negative, and PCV2b-infected pigs thatdied or were euthanized early due to PCVAD (filled circles) were notincluded in the analysis of group means. The (*) indicates significantdifferences on that day.

FIG. 5 illustrates PCV2-specific antibody response in conventionalspecific-pathogen-free pigs vaccinated with the chimeric PCV1-2b virusand subsequently challenged with PCV2a or PCV2b wildtype virus. The meanserum S/P ratio+/−SEM is plotted for each treatment group throughout theexperiment, with (*) indicating significant differences betweenvaccinated (vax) and unvaccinated (PBS) groups. All pigs were challengedwith PCV2a or PCV2b at 56 dpv. Groups with different letters at 77 dpv(or 21 dpc) are significantly different on that day.

FIG. 6 shows a comparison of overall lymphoid lesion scores inconventional SPF pigs vaccinated with PCV1-2b or PBS buffer andsubsequently challenged with PCV2a or PCV2b. Combined lymphoiddepletion, histiocytic replacement, and PCV2-specific antigen scores forthe lymph nodes, spleen, and tonsil were compared between vaccinated andunvaccinated pigs by challenge PCV2 virus subtype. Circles indicatemedian, boxes are 1^(st) and 3rd quartiles, and whiskers indicate therange of data. Pairs of treatments with significantly different.

FIG. 7 illustrates detection and quantification of PCV2 viral DNA loadsin serum and lymphoid tissue samples in conventional pigs vaccinatedwith PBS buffer or PCV1-2b and subsequently challenged with PCV2a orPCV2b. FIG. 7(A) shows a quantification of viremia and viral loads insera using qPCR. Group mean log viral genomic copies/ml of serum+/−SEMis plotted for each treatment group. All samples at 56 dpv (or 0 dpc) aswell as all samples from vaccinated pigs were negative for PCV2a orPCV2b DNA. FIG. 7(B) shows a quantification of viral DNA loads in TBLNtissues using qPCR. Median viral DNA loads in lymphoid tissuesdetermined for each pig are plotted. Circles indicate median, boxes are1^(st) and 3^(rd) quartiles, and whiskers indicate the total range ofdata. Upon challenge with PCV2a or PCV2b, viral DNA was detected in thelymph nodes of all unvaccinated pigs, while only 1/10 vax/PCV2b pigs and5/10 vax/PCV2a pigs had detectable viral DNA. The (*) indicatessignificant differences between vaccinated and unvaccinated pairs, basedon challenge virus subtype.

DETAILED DESCRIPTION OF THE INVENTION

PCV2 infection and PCVAD continue to pose a major threat to global swinepopulations. PCVAD arguably is the most economically-important diseasefacing the swine industry today. Wasting, microscopic lesions oflymphoid depletion with histiocytic infiltration, and the presence ofPCV2 antigen or DNA in the lesions are three characteristic criteria todiagnose PCVAD in a pig (Segales, J. et al. 2005, supra). It is known,however, that not all pigs infected with PCV2 will develop clinicalPCVAD and coinfecting viral and bacterial pathogens are usuallynecessary to induce the full-spectrum of clinical PCVAD (Albina, E. etal., 2001, An experimental model for post-weaning muitisystemic wastingsyndrome (PMWS) in growing piglets, J Comp Pathol 125:292-303; Magar, R.et al., 2000, Experimental transmission of porcine circovirus type 2(PCV2) in weaned pigs: a sequential study, J Comp Pathol 123:258-69).Prior to 2005, the virus detected from PCVAD cases in the United Statesand Canada was almost exclusively of the PCV2a subtype. Consequently,four commercial vaccines all based on the PCV2a subtype were developedand are currently used worldwide in swine herds. The PCV2a strainsisolated worldwide are closely-related and share 95 to 99% nucleotidesequence identities, and thus these commercial vaccines have beeneffective against PCV2a and PCVAD in the global swine populations.

Recently, the outbreaks of more severe PCVAD cases in certain areas inthe United States and Canada have been attributed to the emergence of anew PCV2b subtype (Gagnon, C. A. et al., 2007, supra). The nucleotidesequences between PCV2a and PCV2b subtypes differ by as much as 10% anddistinct amino acid sequence motifs distinguishing the two subtypes havebeen identified (Cheung, A. K. et al., 2007, supra), thus raising thequestion whether or not the current commercial vaccines basedexclusively on PCV2a subtype can fully protect against the new PCV2bsubtype infection. In the past few years, the PCV2 prevalence in theglobal swine herds has shifted to predominantly PCV2b subtype, and infact, the majority of recent PCVAD cases in the United States areassociated with the new PCV2b subtype (Cheung, A. K. et al., 2007,supra; Firth, C et al., 2009, Insights into the evolutionary history ofan emerging livestock pathogen; porcine circovirus 2, J Virol83:12813-21). The PCV2a subtype-based vaccines are still in useworldwide since the PCV2a vaccines have been shown to providecross-protection (Fort, M. et al., 2008, supra; Fort, M. et al. 2009,supra; Opriessnig, T. et al., 2009, supra; Segales, J. et al., 2008,supra). However, the extent of cross-protection offered by thePCV2a-based vaccines against the circulating new PCV2b subtype isunknown, and the global swine industry will certainly be benefited fromhaving access to a vaccine that is based on the currently-circulatingpredominant PCV2b subtype. Therefore, one of the objectives of thepresent invention is to develop a new generation vaccine based on thenew PCV2b subtype, and to evaluate the efficacy of the PCV2b-basedvaccine against both PCV2a and PCV2b challenges.

The present invention provides an attenuated live chimeric virus of PCV1and PCV2. In a particular embodiment, a PCV1 virus expressing a capsidprotein of PCV2 subtype PCV2b is constructed. As an exemplary process,the inventors first generated an infectious DNA clone of PCV2b subtype,and then constructed a novel chimeric virus, PCV1-2b, containing theimmunogenic capsid gene of the PCV2b subtype in the genomic backbone ofthe non-pathogenic PCV1. The pathogenicity and immunogenicity of thenovel chimeric PCV1-2b virus were first evaluated in CD/CD pigs.Subsequently, a challenge and cross-challenge study was performed inconventional pigs to determine the vaccine efficacy of the PCV1-2bchimeric vaccine virus. The inventors demonstrated that the chimericPCV1-2b vaccine virus is attenuated in pigs and induces protectiveimmunity against PCV2b and cross-protective immunity against PCV2a.Therefore. this new chimeric PCV1-2b virus should be an excellentcandidate as a live-attenuated vaccine against both PCV2b and PCV2ainfections and PCVAD.

The following examples demonstrate certain aspects of the presentinvention. However, it is to be understood that these examples are forillustration only and do not purport to be wholly definitive as toconditions and scope of this invention. It should be appreciated thatwhen typical reaction conditions (e.g., temperature, reaction times,etc.) have been given, the conditions both above and below the specifiedranges can also be used, though generally less conveniently. Theexamples are conducted at room temperature (about 23° C. to about 28°C.) and at atmospheric pressure. All parts and percents referred toherein are on a weight basis and all temperatures are expressed indegrees centigrade unless otherwise specified.

Example 1 PCV1 and PCV2 Virus Isolates

The PCV1 infectious DNA clone was constructed in previous studies andshown to be non-pathogenic in pigs (Fenaux, M. et al., 2002, Clonedgenomic DNA of type 2 porcine circovirus is infectious when injecteddirectly into the liver and lymph nodes of pigs: characterization ofclinical disease, virus distribution, and pathologic lesions, J Virol76:541-51; Fenaux, M. et al. 2004A, supra; Fenaux, M. et al., 2003,supra). PCV2a isolate ISU-40895 (SEQ ID No:1, Genbank accession no.AF264042) was recovered from a pig with PCVAD in an Iowa farm in 1998(Fenaux, M. et al., 2000, supra) and has been used extensively in PCV2pathogenicity studies (Fenaux, M. et al., 2002, supra; Fenaux, M. etal., 2004, supra; Fenaux, M. et al., 2003, supra; Opriessnig, T. et al.,2006, Evidence of breed-dependent differences in susceptibility toporcine circovirus type-2-associated disease and lesions, Vet Pathol43:281-93; Opriessnig, T. et al., 2006, Effects of the timing of theadministration of Mycoplasma hyopneumoniae bacterin on the developmentof lesions associated with porcine circovirus type 2, Vet Rec158:149-54; Opriessnig, T. et al., 2004, Experimental reproduction ofpostweaning muitisystemic wasting syndrome in pigs by dual infectionwith Mycoplasma hyopneumoniae and porcine circovirus type 2, Vet Pathol41:624-40; Opriessnig, T. et al., 2003, Effect of vaccination withselective bacterins on conventional pigs infected with type 2 porcinecircovirus, Vet Pathol 40:521-9). PCV2a-40895 is capable of causingPCVAD microscopic lesions and clinical disease in experimentalconditions (Opriessnig, T. et al., 2006, supra; Opriessnig, T. et al.,2004, Effect of porcine parvovirus vaccination on the development ofPMWS in segregated early weaned pigs coinfected with type 2 porcinecircovirus and porcine parvovirus, Vet Microbiol 98:209-20; Opriessnig,T. et al., 2004, supra). The PCV2b strain used in the study wasconfirmed to be an authentic PCV2b subtype by sequencing of the entireviral genome (SEQ ID No:2, Genbank accession no. GU799576). The genomicDNA of the PCV2b was used as the source for the construction of theinfectious DNA clones of PCV2b as well as the chimeric PCV1-2binfectious DNA clone. The pathogenicity of the PCV2b virus was notdetermined, prior to the present invention, in experimental infections.

Example 2 Generation of Infectious DNA Clones of PCV2b and ChimericPCV1-2b

The method for the construction of the infectious DNA clone ofPCV2a-40895 has been reported previously (Fenaux, M. et al., 2002,supra), and a similar approach was used in the present invention toproduce an infectious DNA clone of PCV2b (FIG. 1 a). Briefly, thefull-length genome of PCV2b was amplified by PCR using a pair of primersA (SEQ ID No:3) and B (SEQ ID No:4) (Table 1) with an overlapping regioncontaining the Sac II restriction enzyme site that is present in allPCV2 strains. The PCR product was then digested with Sac II (New EnglandBiolabs) and ligated into pBluescript II SK(+) (pBSK+) (Stratagene) toproduce an infectious DNA clone of PCV2b.

To produce the chimeric PCV1-2b infectious DNA clone, overlap-extensionPCR was used to replace the PCV1 capsid gene in the backbone of a PCV1infectious clone with the capsid gene from PCV2b (FIG. 1 b). Thefull-length chimeric PCV1-2b genome was assembled from three overlappingPCR fragments (Table 1). Each amplicon was generated using Platinum TaqHiFi mastermix (Invitrogen) with the same amplification parameters (95°C. 3 min.; 40 cycles of 95° C. 30 sec, 55° C. 30 sec, 68° C. 1 min). PCRproducts were purified using the QIAquick Gel Extraction Kit (Qiagen).Fusion PCRs consisting of two steps were used to assemble the chimericPCV1-2b DNA clone: an assembly reaction without primers using 50 ng ofeach fragment as template (20 cycles of 95° C. 30 sec, 55° C. 30 sec,68° C. 1 min) followed by amplification using outer primers (40 cyclesof 95° C. 30 sec, 55° C. 30 sec, 68° C. 1 min). The 1,043 bp fragmentamplified from a PCV1 clone with primers G (SEQ ID No:9) and H (SEQ IDNo:10) (Table) was first fused with the 718 bp fragment containing theentire capsid gene amplified from PCV2b with primers C (SEQ ID No:5) andD (SEQ ID No:6) (Table 1). The 122 bp fragment amplified from PCV1 withprimers E (SEQ ID No:7) and F (SEQ ID No:8) (Table 1) was then added,resulting in a complete chimeric PCV1-2b genome flanked by Kpn Irestriction sites. The chimeric fusion product was subsequently digestedwith Kpn I (New England Biolabs), and cloned into pBSK+. Monomeric DNAclones were completely sequenced to confirm that no unwanted mutationshad been introduced during PCR amplification steps.

Example 3

Dimerization of PCV2b and PCV1-2b DNA Clones

Previous studies showed that dimerized PCV2a clones with two copies offull-length PCV2a genome ligated head-to-tail in tandem are moreefficient in generating infectious virus in both in vitro transfectionin PK-15 cells and in vivo transfection in pigs (Fenaux, M. et al.,2002, supra; Fenaux, M. et al., 2004A, supra; Fenaux, M. et al., 2003,supra). Therefore, in the present invention both PCV2b and PCV1-2bclones were dimerized to produce more robust and efficient infectiousclones. Briefly, plasmid DNAs containing PCV2b and PCV1-2b monomericgenomes were extracted using the QIAprep Spin Miniprep kit (Qiagen). Thepurified plasmid DNA was linearized using Sca I (New England Biolabs)and subjected to partial digestion with Kpn I by incubation at 37° C.for 30 sec to generate two fragments of approximately 3,100 and 3,700 bpwhich were then purified by gel extraction. The two fragments werecombined and ligated using T4 DNA ligase (Promega) to generate thetandem dimerized infectious DNA clones for both PCV2b and PCV1-2b.

Example 4 Viability Testing of PCV2b and PCV1-2b Infectious DNA Clonesin PK-15 Cells

The viability and infectivity of the PCV2b and chimeric PCV1-2b DNAclones were tested in vitro after transfection using an indirectfluorescent assay (IFA) as previously described (Fenaux, M. et al.,2002, supra). Briefly, 6.5 μg of each dimerized DNA clone was added to1,250 μl OPTIMEM media (Invitrogen) and 6.25 μl PLUS Reagent(Invitrogen) and incubated at room temperature for 5 min. After additionof 16 μl Lipofectamine LTX (Invitrogen), the mixture was incubated atroom temperature for 30 min., followed by addition of 250 μl OPTIMEMmedia. T25 cell culture flasks (Corning) containing PK-15 cells at60-70% conflueney were washed with MEM media (Invitrogen), thetransfection mixture was added, and the flasks were then incubated for 6hrs at 37° C. After incubation, 8 ml growth media (MEM containing 10%fetal bovine serum and 2× antibiotic/antimycotic solution [Invitrogen])was added to each flask and incubated for additional 72 hrs at 37° C.The transfected cells in each T25 flasks were then frozen and thawedthree times at −80° C., and cell lysates were centrifuged at 2,500×g at4° C. for 10 min to remove cellular debris. The supernatants wereharvested and used to infect fresh PK-15 cells seeded in 48-well plates(BD-Falcon) at 50% confluency. After addition of 100 μl of transfectionsupernatant per well, plates were incubated for 1 hr at 37° C., followedby addition of 500 μl growth media to each well and incubation for 72hrs at 37° C. Capsid proteins in the nuclei of infected PK-15 cells werevisualized using IFA as previously described (Fenaux, M. et al. 2002,supra). Briefly, cells were fixed using 80% acetone in PBS at 4° C. for30 min, washed one time with PBS buffer, and incubated with a 1:1,000dilution of a PCV2 mono-specific mouse monoclonal antibody (RuralTechnologies, Inc.: Brookings, S. Dak.) at 37° C. for 45 min. Afterwashing three times with PBS buffer, the cells were incubated with a1:50 dilution of FITC-labeled secondary goat anti-mouse IgG (KPL) at 37°C. for 45 min. After washing with PBS buffer, the cells were thencovered with Fluoromount G (Southern Biotech) and examined under afluorescent microscope.

PCV2b and chimeric PCV1-2b DNA clones are infectious when transfectedinto PK-15 cells: Full-length single copy and tandemly-dimerized DNAclones of PCV2b and chimeric PCV1-2b were constructed and verified byfull-length sequencing. Transfection of PK-15 cells with dimers of bothDNA clones resulted in the production of infectious progeny virions asdetected by IFA with PCV2 capsid-specific monoclonal antibodies. Theinfectious titers of both PCV2b and chimeric PCV1-2b virus stocks wereapproximately 10^(4.5) TCID₅₀/ml.

Example 5 Generation and Titration of Infectious PCV2a, PCV2b, andPCV1-2b Virus Stocks

To prepare inocula for the in vivo pig studies, infectious virus stockswere generated for PCV2a-40895, PCV2b, and PCV1-2b by transfection ofPK-15 cells in T25 flasks with dimerized infectious DNA clones (seeabove) (Fenaux, M. et al. 2002.supra; Fenaux, M. et al. 2003, supra).The titration of these infectious virus stocks by IFA was performedessentially as described previously (Fenaux, M. et al. 2002, supra).Briefly, PK-15 cells were seeded in 48-well plates (BD-Falcon) at 60%confluency and incubated for 3 his at 37° C. Serial ten-fold dilutionsof each of the virus stocks were produced in MEM, and each dilution wasinoculated onto four separate wells with 100 μl per well. Plates wereincubated for 1 hr at 37° C., followed by addition of 500 μl growthmedia to each well and continued incubation for 72 his at 37° C.Positive signals in the nuclei of infected cells were visualized in eachwell using IFA (see above). The 50% tissue culture infective dose(TCID₅₀) per ml was calculated according to the method of Reed & Muench.

Example 6 Experimental Design for the Pathogenicity Study of PCV2b andChimeric PCV1-2b in Caesarean-Derived Colostrum-Deprived (CD/CD) Pigs

CD/CD pigs are considered to be a superior model system for the study ofPCV2 pathogenicity since characteristic pathological lesions andclinical PCVAD can be reproduced in this model (Allan, G. et al., 2003,supra; Bolin, S. R. et al., 2001, supra; Harms, P. A. et al., 2001,supra; Kennedy, S. et al., 2000, supra; Tomas, A. et al., 2008, supra).To determine the pathogenicity of the chimeric PCV1-2b virus and compareit to the wildtype PCV2b virus, a total of 30 CD/CD pigs (Struve Labs,Manning, Iowa), approximately 9 weeks of age, were randomly assigned tothree groups in rooms of 10 animals each. Prior to inoculation, each pigwas weighed, bled, and confirmed to be negative for the presence of PCV2antibodies. Group 1 pigs were each mock-inoculated with 3 ml PBS buffer(2 ml intranasally and 1 ml intramuscularly) and served as uninfectedcontrols. Pigs in group 2 were each inoculated with 3 ml of inoculumcontaining 2×10⁴⁵′ TCID₅₀ chimeric PCV1-2b virus (2 ml intranasally and1 ml intramuscularly). Pigs in group 3 were each similarly inoculatedwith 2×10^(4.5) TCID₅₀ wildtype PCV2b virus. Blood samples werecollected prior to inoculation, and weekly thereafter from each piguntil necropsy at 21 or 42 days post-inoculation (dpi). At 21 dpi, fiverandomly assigned pigs from each group were necropsied. The remainingfive pigs in each group were necropsied at 42 dpi.

Example 7 Experimental Design for the Pathogenicity Study of PCV2b andChimeric PCV1-2b in Caesarean-Derived Colostrum-Deprived (CD/CD) Pigs

CD/CD pigs are considered to be a superior model system for the study ofPCV2 pathogenicity since characteristic pathological lesions andclinical PCVAD can be reproduced in this model (Allan, G. et al., 2003,supra; Bolin, S. R. et al., 2001, supra; Harms, P. A. et al., 2001,supra; Kennedy, S. et al. 2000, supra; Tomas, A. et al. 2008, supra). Todetermine the pathogenicity of the chimeric PCV1-2b virus and compare itto the wildtype PCV2b virus, a total of 30 CD/CD pigs (Struve Labs,Manning, Iowa), approximately 9 weeks of age, were randomly assigned tothree groups in rooms of 10 animals each. Prior to inoculation, each pigwas weighed, bled, and confirmed to be negative for the presence of PCV2antibodies. Group 1 pigs were each mock-inoculated with 3 ml PBS buffer(2 ml intranasally and 1 ml intramuscularly) and served as uninfectedcontrols. Pigs in group 2 were each inoculated with 3 ml of inoculumcontaining 2×10^(4.5,) TCID₅₀ chimeric PCV1-2b virus (2 ml intranasallyand 1 ml intramuscularly). Pigs in group 3 were each similarlyinoculated with 2×10 TCID₅₀ wildtype PCV2b virus. Blood samples werecollected prior to inoculation, and weekly thereafter from each piguntil necropsy at 21 or 42 days post-inoculation (dpi). At 21 dpi, fiverandomly assigned pigs from each group were necropsied. The remainingfive pigs in each group were necropsied at 42 dpi.

Chimeric PCV1-2b virus is attenuated in CD/CD pigs whereas the wildtypePCV2b virus induces pathological lesions and clinical diseasescharacteristic of PCVAD: To definitively assess the pathogenic potentialof the chimeric PCV1-2b virus, the pathogenicity study was, conducted ina CD/CD pig model which has been shown to be the most reproducible andhighly sensitive PCVAD disease model (Allan, G. et al., 2003, supra;Bolin, S. R. et al., 2001, supra; Harms, P. A. et al., 2001, supra;Kennedy, S. et al. 2000, supra).

Clinical Signs and Gross Lesions:

CD/CD pigs experimentally-inoculated with chimeric PCV1-2b virus or PBSbuffer had no apparent clinical signs of PCVAD throughout the study,whereas experimental inoculation of CD/CD pigs with wildtype PCV2bresulted in the PCVAD-associated death or early euthanasia in four ofthe 10 PCV2b-infected pigs: one pig died at 18 dpi, another died at 27dpi, and two other pigs had to be euthanized at 34 dpi due toprogressive weight loss.

Pigs from each of the three treatment groups had similar weight gainthrough 21 dpi, but the wildtype PCV2b-infected pigs had a decrease inweight gain in four of the five pigs after 21 dpi (three of which diedor were euthanized early due to weight loss). Macroscopic lung lesionswere not observed in pigs inoculated with the chimeric PCV1-2b virus,whereas the three PCV2b-inoculated pigs necropsied at 27 dpi and 34 dpidisplayed moderate lung lesions. Lymph nodes were enlarged in bothPCV1-2b and PCV2b infected pigs compared to the PBS controls. However,the number of pigs with enlarged lymph nodes and the magnitude ofenlargement was less in PCV1-2b-infected pigs than in wildtype PCV2bpigs (data not shown).

Microscopic Lesions:

Microscopic lesions were analyzed by treatment in two groups: all pigsnecropsied at 21 dpi or before (including the PCV2b-infected pig thatdied at 18 dpi), and all pigs necropsied at 42 dpi (including the threePCV2b-infected pigs that died or were euthanized at 27 dpi and 34 dpi).

Microscopic lesions in lung, liver, thymus, heart, kidney, ileum, colon,lymph nodes, spleen, and tonsil are summarized in Table 2. As expected,there were no remarkable microscopic lesions in pigs inoculated with PBSbuffer. Characteristic PCV2-associated microscopic lesions in lymphoidtissues in pigs inoculated with chimeric PCV1-2b virus were decreased inincidence and severity compared to the pigs experimentally inoculatedwith the wildtype PCV2b at both 21 and 42 dpi (Table 2). Microscopiclesions in other non-lymphoid tissues included mild to severeinfiltration with lymphocytes and macrophages (Table 2). Lesions in thesmall and large intestine were found almost exclusively in wildtypePCV2b-infected pigs.

The mean group overall lymphoid lesion score in pigs inoculated with thechimeric PCV1-2b virus was significantly (p=0.045) lower than that inpigs inoculated with the wildtype PCV2b virus and was not different frompigs inoculated with PBS buffer at 21 dpi (FIG. 3).

PCV2 Serology:

Serum samples taken from pigs that died or were euthanized early wereanalyzed along with those from the next scheduled necropsy (e.g. serumfrom the pig that died at 18 dpi was included in the analysis of samplesfrom 21 dpi necropsy). PCV2 capsid-specific IgG antibodies were detectedin the sera of some PCV1-2b-infected pigs as early as 7 dpi, withseroconversion in 9/10 pigs by 21 dpi (FIG. 2). The PCV2 IgG antibodytiters in chimeric PCV1-2b virus-inoculated pigs plateaued by 28 dpi andremained high through the end of the study at 42 dpi. In pigsexperimentally-inoculated with wildtype PCV2 virus, the seroconversionwas less uniform: only 3/10 pigs were seropositive by 21 dpi, and 5/10pigs had no detectable seroconversion in the study. However, only 2/5PCV2b-infected pigs survived to the scheduled necropsy at dpi 42, andboth of them did seroconvert at 28 dpi, with increasing IgG antibodytiters towards the end of the study. Of the pigs that died or wereeuthanized early due to clinical PCVAD, only 1/4 had detectablePCV2-specific antibody at any time, and none had detectable serumanti-PCV2 antibody on the day they were necropsied. No PCV2capsid-specific antibodies were detected in any of the PBSbuffer-inoculated pigs throughout the study.

Prevalence and Amount of PCV2-Specific Antigen in Tissues by IHC:

Prevalence and mean group amounts of PCV2 antigen in the differenttissues are summarized in Table 3. All pigs necropsied at 21 dpi orbefore (including the PCV2b-infected pig that died at 18 dpi) wereanalyzed together, and all pigs necropsied at 42 dpi (including thethree PCV2b-infected pigs that died or were euthanized at 27 dpi and 34dpi) were analyzed together. In general, the incidence and amount ofPCV2 antigen were less in pigs inoculated with the chimeric PCV1-2bvirus compared to the pigs inoculated with the wildtype PCV2b virus(Table 3). There was a single PCV1-2b-inoculated pig that accounted forthe positive results in all 21 dpi tissues except the tonsil, where fourof five pigs were positive. Tissues from PCV1-2b-inoculated pigs weremostly negative at 42 dpi, with the exception of the lymph nodes, inwhich low amounts of PCV2 antigen were detected in four of five pigs.

PCV2 Viremia and Serum Viral Loads:

The PCV2 viral load in the sera of infected animals was determined usinga modified qPCR assay that amplifies part of the PCV2b capsid gene andthe qPCR assay is known to work with both PCV2b and PCV1-2b (Yang, Z. Z.et al., 2007, Detection of PCV2 DNA by SYBR Green 1-based quantitativePCR. J Zhejiang Univ Sci B 8:162-9). All serum samples taken prior toinoculation at day 0 and from PBS buffer-inoculated pigs throughout thestudy were confirmed to have no detectable PCV2 DNA. Serum samples takenfrom pigs that died or were necropsied early due to PCVAD disease wereanalyzed along with those from the next scheduled necropsy (e.g. serumfrom pig that died at 18 dpi was included in the analysis of samplesfrom 21 dpi).

The serum viral loads present in pigs inoculated with the chimericPCV1-2b virus were significantly (p<0.009) lower than the serum viralloads in PCV2b-inoculated pigs from 14 dpi through the end of the study(FIG. 4 a). The mean serum viral loads peaked at 10⁷ genomic copies/mlat 14 dpi in PCV1-2b-infected pigs, and steadily decreased towards theend of the study. The maximum value achieved for any individualPCV1-2b-infected pig was 10⁸ genomic copies/ml at 14 dpi. In contrast,the mean serum viral loads in PCV2b-infected pigs remained above 10⁸genomic copies/ml from 14 dpi until after 28 dpi, with 10¹² being themaximum value achieved by an individual pig. The four pigs that diedearly or were euthanized due to PCVAD had higher levels of serum viralloads than the other PCV2b-inoculated pigs. After 7 dpi, PCV2b-infectedpigs had at least ten-fold greater viral genomic copies per ml of serumthan the PCV1-2b-infected pigs, and one hundred-fold greater at 21 dpiand 28 dpi.

PCV2 Viral Load in Lymphoid Tissues:

The amount of PCV2 viral DNA in the TBLN tissues collected at necropsyof each pig was determined using the same qPCR assay as above. All TBLNtissue samples from PBS buffer-inoculated pigs were confirmed to benegative for PCV2 DNA. In order to enable a meaningful comparison ofgroup mean viral loads at 21 dpi and 42 dpi, pigs that died early orwere euthanized due to PCVAD disease were tested but not included in theanalysis. The viral load present in each mg of TBLN tissue was found tobe significantly lower in PCV1-2b-inoculated pigs than inPCV2b-inoculated pigs at 21 dpi (p=0.005) (FIG. 4 b). Pigs that died orwere euthanized early due to PCVAD had the highest viral loads in TBLNtissues among all that were tested.

Example 8 Experimental Design for Vaccination and Immunogenicity Studyof the Chimeric PCV1-2b Virus in Conventional Specific-Pathogen-Free(SPF) Pigs

A total of 40, 3-week-old, cross-breed SPF pigs were purchased from acommercial farm (Virginia Tech Swine Center, Blacksburg, Va.) that isknown to be free of PCV, PRUSV, PPV, M. hyopneumonia, and swinehepatitis E virus. The piglets were randomly assigned to two groups of20 pigs each, and housed separately. Prior to inoculation, each pig wasweighed, bled, and confirmed to be negative for PCV2 antibodies. Group 1pigs were each vaccinated intramuscularly (IM) with 1 ml of the chimericPCV1-2b virus (10^(3.5) TCID₅₀ infectious virus per pig). Group 2 pigswere each mock-vaccinated IM with 1 ml PBS buffer and served asunvaccinated controls. All animals were monitored daily for clinicalsigns, and blood samples were collected prior to inoculation, and weeklythereafter from each pig through 56 days post-vaccination (dpv).

Example 9 Experimental Design for Challenge and Cross-Challenge ofVaccinated Pigs with Wildtype PCV2b Subtype and PCV2a Subtype,Respectively

At 56 dpv, the 20 vaccinated pigs were further divided into two groupsof 10 pigs each and housed in separate rooms: 10 vaccinated pigs wereeach challenged with 2×10⁴⁵′ TCID₅₀ wildtype PCV2b virus (2 mlintranasally and 1 ml IM), and the other 10 vaccinated pigs were eachsimilarly cross-challenged with 2×10^(4.5,) TCID₅₀ wildtype PCV2a virus.The 20 unvaccinated control pigs were also further divided into twogroups of 10 pigs each, and similarly inoculated with PCV2b and PCV2a,respectively. Blood samples were collected weekly through 21 dayspost-challenge (dpc) (or 77 dpv), at which time all pigs werenecropsied.

Vaccination with chimeric PCV1-2b virus confers protective immunity inconventional pigs against challenge with PCV2b subtype andcross-challenge with PCV2a subtype: Since the chimeric PCV1-2b vaccineis intended for usage in conventional pigs in the field, the inventorsthus conducted this vaccine efficacy and challenge study usingconventional SPF pigs.

Clinical Signs and Gross Lesions:

None of the pigs developed clinical signs consistent with PCVADthroughout the study, and no difference in weight gain was observedbetween treatment groups. A single pig assigned to theunvaccinated/PCV2b challenge group died at 2 dpc from bacterialsepticemia unrelated to PCVAD, and was not included in the analysis.Upon necropsy, macroscopic lung lesions were not observed in any of thetreatment groups. Mild to moderately enlarged lymph nodes were seen inall treatment groups, with no significant differences observed betweentreatments.

Serology:

All pigs were determined to be negative for antibodies against PCV2,PRRSV, PPV, and SIV prior to the study. PCV2 capsid-specific antibodieswere detected in the sera of PCV1-2b-vaccinated pigs as early as 14 dpv,with seroconversion occurred in 15/20 pigs at 21 dpv and 20/20 by 28 dpv(FIG. 5). The PCV2 IgG antibody titers plateaued by 35 dpv and remainedhigh at the time of challenge at 56 dpv. Antibodies to PCV2 were notdetected in unvaccinated control pigs until after challenge at 14 dpc(FIG. 5): 10/10 PCV2a-challenged pigs were seropositive, compared toonly 2/9 seropositive PCV2b-challenged pigs at 14 dpc. By 21 dpc, 7/9unvaccinated PCV2b-challenged pigs had seroconverted.

Microscopic Lesions:

After challenge, the incidence and severity of microscopic lesion scoresin lymphoid tissues were higher in unvaccinated pigs compared to thevaccinated ones (Table 4). In general, the characteristic lymphoiddepletion and histiocytic replacement scores were lower in vaccinatedpigs compared to the unvaccinated controls (Table 4). The overalllymphoid lesion score in vaccinated pigs was significantly lower thanthat in unvaccinated pigs for both PCV2a (p=0.0001) and PCV2b (p-0.04)challenge groups (FIG. 6).

Amount of PCV2 Antigen by IHC in Tissues:

IHC was used to detect PCV2-specific antigens in each tissue type (Table4). Similarly to the histological lesions, in general the incidence andthe amount of PCV2 antigen in tissues were reduced in vaccinated pigscompared to unvaccinated controls (Table 4).

Viremia and Serum Viral Load:

The amounts of PCV2a or PCV2b viral DNA in the serum after challengewere quantified using a modified qPCR assay that is specific for thedetection of PCV2a or PCV2b rep gene but is not capable of amplifyingthe vaccine virus PCV1-2b (Mcintosh, K. A. et al. 2009. Development andvalidation of a SYBR green real-time PCR for the quantification ofporcine circovirus type 2 in serum, buffy coat, feces, and multipletissues. Vet Microbiol 133:23-33). Serum samples collected prior tochallenge at 56 dpv were confirmed to have no detectable PCV2 DNA. Therewas no detectable PCV2a or PCV2b viremia in vaccinated pigs at any timepoint post-challenge. In contrast, PCV2a or PCV2b viremia was detectedin all unvaccinated pigs at every time point post-challenge (FIG. 7 a).There was no statistically significant difference in serum viral loadbetween unvaccinated pigs challenged with PCV2a or PCV2b virus.

PCV2a or PCV2b Viral Load in Lymphoid Tissues:

The amounts of PCV2a or PCV2b viral DNA in the TBLN tissues collected atnecropsy are summarized in FIG. 7 b. The viral loads in each mg of TBLNtissues were found to be significantly lower in vaccinated pigs comparedto unvaccinated ones in both PCV2a (p-0.0001) and PCV2b (p<0.0001)challenge groups (FIG. 7 b). Only 1/10 of the vaccinated and PCV2bchallenged pigs had detectable PCV2b viral DNA in the TBLN tissues, witha viral load of 10⁵ genomic copies/mg. Five of ten vaccinated and PCV2achallenged pigs had detectable PCV2a viral DNA in the TBLN tissues, eachwith a viral load of 10⁵-10⁶ genomic copies/mg. The viral load in theTBLN tissues of the unvaccinated pigs ranged from 10⁸-10¹⁰ genomiccopies/mg, regardless of PCV2a or PCV2b challenge.

Experimental Materials and Methods

Cells: A subclone of the PK-15 cell line that is free of PCV1contamination was produced previously by end-point dilution of the PK-15cells (ATCC CCL-33) (Fenaux, M. et al., 2000, supra; Fenaux, M. et al.,2002, supra). The PCV1-free PK-15 cell line was used for the generationof infectious virus stocks, and for the infectivity titration of thevirus stocks used in the present invention.

Serology: ELISA was used to detect anti-PCV2 IgG in each serum sample aspreviously described (Nawagitgul, P. et al., 2002, Modified indirectporcine circovirus (PCV) type 2-based and recombinant capsid protein(ORF2)-based enzyme-linked immunosorbent assays for detection ofantibodies to PCV, Clin Diagn Lab Immunol 9:33-40). Serum samples withsample: positive (S:P) ratios greater than 0.2 were considered to bepositive for anti-PCV2 antibodies. All pigs were confirmed to be PCV2seronegative by ELISA prior to the start of the animal experiments.

Clinical evaluation: Following inoculation, vaccination or challenge,pigs were evaluated daily for clinical signs of PCVAD including wasting,respiratory distress, and behavioral changes such as lethargy andinappetance.

Gross pathology and histopathology: Necropsies were performed at thedesignated time for the pathogenicity (dpi 21 and 42) or challengeexperiment (dpc 21 or dpv 77) on all pigs in a blinded fashion.Estimates of macroscopic lung lesions (ranging from 0-100% of the lungaffected) and lymph node size (ranging from 0 [normal] to 3 [four timesthe normal size]) were made and scored for each pig (Halbur, P. G. etal., 1995, Comparison of the pathogenicity of two US porcinereproductive and respiratory syndrome virus isolates with that of theLelystad virus, Vet Pathol 32:648-60; Opriessnig, T. et al., 2004,supra).

Sections of lung, lymph nodes (superficial inguinal, mediastinal,tracheobronchial, and mesenteric), tonsil, heart, thymus, ileum, kidney,colon, spleen, and liver were collected during each necropsy and fixedin 10% neutral-buffered formalin and processed routinely forhistological examination and immunohistochemistry (IHC). Also, samplesof tracheobronchial lymph node (TBLN) were collected from each pig forDNA extraction and quantification of viral genomes by real-time PCR.Microscopic lesions in the lungs, heart, liver, kidney, ileum, and colonwere scored in a blinded manner, as described previously (Opriessnig, T.et al., 2004, supra). Lymphoid tissues including lymph nodes, spleen,and tonsil were evaluated based on lymphoid depletion and histiocyticreplacement of follicles, ranging from 0 (normal) to 3 (severe)(Opriessnig, T. et al., 2004, supra).

Immunohistochemistry (IHC): IHC for detection of PCV2-specific antigenwas performed on formalin-fixed, paraffin-embedded sections of lung,lymph nodes (superficial inguinal, mediastinal, tracheobronchial, andmesenteric), tonsil, heart, thymus, ileum, kidney, colon, spleen, andliver using a rabbit polyclonal antiserum (Sorden, S. D. et al. 1999.Development of a polyclonal-antibody-based immunohistochemical methodfor the detection of type 2 porcine circovirus in formalin-fixed,paraffin-embedded tissue. J Vet Diagn Invest 11:528-30). The scores ofPCV2 antigen in each tissue were estimated in a blinded fashion and thescores ranged from 0 (no antigen) to 3 (greater than 50% lymphoidfollicles contained cells with positive PCV2 antigen staining inlymphoid tissues or high amount of PCV2 antigen in other tissuesections) (Opriessnig, T. et al., 2004, supra).

Overall microscopic lymphoid lesion scores: The average scores of theoverall microscopic lymphoid lesions were calculated for each pig asdescribed previously (Opriessnig, T. et al., 2004, supra). These lesionscores are based on the combined lymphoid depletion (LD), histiocyticreplacement (HR), and PCV2 antigen present in the lymphoid tissues asdetermined by IHC.

Quantitative real-time PCR to determine viral DNA loads: Total DNA wasextracted from serum samples using the QIAamp DNA minikit (Qiagen Inc)according to the “blood and body fluids” protocol supplied by themanufacturer. TBLN tissues collected during necropsies were homogenizedto produce a 10% tissue homogenate suspension in sterile PBS buffer, andtotal DNA was extracted using the QIAamp DNA minikit with the “tissue”protocol supplied by the manufacturer (Qiagen Inc). All TBLN DNAextracts were diluted at least 1:100 in sterile H₂O in order toeliminate background fluorescence from SYBR green binding to porcinegenomic DNA. Due to the extremely high viral DNA concentrations in somesamples, some serum and TBLN extracts were diluted as much as 1:10⁶ inorder to bring them within the linear range of qPCR detection. Two SYBRgreen I-based qPCR assays were modified for use in the present inventionto quantify the PCV2 genomes present in TBLN and serum DNA extracts(Mcintosh, K. A. et al., 2009, Development and validation of a SYBRgreen real-time PCR for the quantification of porcine circovirus type 2in serum, buffy coat, feces, and multiple tissues, Vet Microbiol133:23-33; Yang, Z, Z. et al., 2007, supra).

In the CD/CD pig pathogenicity study, the inventors utilized apreviously published qPCR assay that amplifies part of PCV2b capsid geneto quantify viral genomes present in serum and TBLN (Yang, Z. Z. et al.,2007, supra).

The protocol was modified to enable the use of the Sensimix SYBR andfluorescein kit (Quantace). Each 25 μl reaction contained 200 nM eachprimer (P1 (SEQ ID No:11): 5′-ATAACCCAGCCCTTCTCCTACC-3′); P2 (SEQ IDNo:12): 5′-GGCCTACGTGGTCTACATTTCC-3′), 200 μM dNTP, 3 mM MgCl₂, and 5 μlDNA extract. Triplicate reactions were run for each sample in MylQ qPCRthermocycler (BioRad) using a modified program (95° C. 10 min.; 35cycles of 95° C. 15 sec, 59° C. 30 sec, 72° C. 30 sec). Viral genomeswere quantified against duplicate standards of PCV2b infectious DNAclone using the relative C₁ method, with a 6-log linear range ofquantification (10³⁻10⁸ copies).

In the challenge and cross-challenge conventional SPF pig study, theinventors used a previously published qPCR assay that amplifies a highlyconserved region of PCV2 replicase gene region to quantify the amountsof viral genomes present in serum and TBLN tissues (Mcintosh, K. A. etal., 2009, supra). This assay does not detect the chimeric PCV1-2bvaccine virus used in the vaccination, and thus allowing for accuratequantification of the challenge PCV2a or PCV2b viruses only. Thisprotocol was modified to enable the use of the Sensimix SYBR andfluorescein kit. Each 25 μl reaction contained 200 nM each primer(PCV2-83F (SEQ ID No:13): 5′-AAAAGCAAATGGGCTGCTAA-3′; PCV2-83R (SEQ IDNo:14): 5′-TGGTAACCATCCCACCACTT-3′), 200 μM dNTP, 5 mM MgCl₂, and 5 DNAextract. Triplicate reactions for each sample were run in a MylQ qPCRthermocycler using a modified program (95° C. 10 min.; 35 cycles of 95°C. 15 sec, 60° C. 15 sec, 72° C. 15 sec). Viral DNA genomes werequantified against duplicate standards of PCV2b infectious DNA cloneusing the relative C₁ method, with a 5-log linear range ofquantification (5×10²-5×10⁶ copies).

Sequence confirmation of virus detected by qPCR: TBLN tissues DNAextracts from selected pigs of each group were tested to confirm thevirus detected by qPCR was the same virus that was inoculated into pigsat the start of each study. PCV1-2b in the CD/CD pathogenicity study wasconfirmed by PCR amplification and partial sequencing using primersspecific for PCV1-2b (PCV2F (SEQ ID No:15): 5′-TGTTGAAGATGCCATTTTTCC-3′;PCV1R (SEQ ID No:16): 5′-GAGGAGTTCTACCCTCTTCC-3′). PCV2a and PCV2b wereamplified and sequenced using primers specific for PCV2 (PCV2F (SEQ IDNo:17): 5′-TGTTGAAGATGCCATTTTTCC-3′; PCV2R (SEQ ID No:18):5′-GAGGTGTTCGTCCTTCCTCA-3′).

Statistical Analysis: Serology and serum qPCR were analyzed usingrepeated measures analysis of variance (ANOVA). TBLN qPCR from the CD/CDpathogenicity study was analyzed using simple ANOVA. For all the ANOVAmodels simple effects comparisons were investigated using the sliceoption of the Glimmix procedure followed by Tukey's procedure formultiple comparisons. TBLN qPCR results from the challenge study wereanalyzed using the exact Wilcoxon 2-sample test. Scores from lymphoidlesions, histopathology lesions, and IHC from both experiments wereassessed with the exact Kruskal-Wallis one way ANOVA, followed by theexact Wilcoxon 2-sample test for the 2-way comparisons of interest. The2-way comparisons were adjusted for multiple comparisons usingBonferroni's procedure. Statistical significance was set to alpha=0.05.All analyses were performed using commercially available software (SASversion 9.2, Cary, N.C., USA).

The inventors have previously developed an inactivated vaccine, SuvaxynPCV2® One Dose™, based on a chimeric virus made from the PCV2a subtype(Fenaux, M. et al. 2004, supra; Fenaux, M. et al. 2003, supra) and thiskilled vaccine has been effective and is currently available on theglobal market. By using a similar strategy, in this present study theinventors first developed a novel chimeric virus PCV1-2b with the capsidgene of the PCV2b subtype cloned in the genomic backbone of thenon-pathogenic PCV1. The use of this chimeric PCV1-2b virus as alive-attenuated new generation vaccine against PCV2 and PCVAD wasthoroughly investigated in CD/CD and conventional pig models.

For use of the chimeric PCV1-2b virus as a live-attenuated vaccine, itis important to ascertain that the vaccine virus is attenuated. ThoughPCV2 alone rarely causes full-spectrum clinical disease in experimentalmodels, the use of CD/CD pigs has resulted in successful reproduction ofclinical PCVAD (Allan, G. et al., 2003, supra; Bolin, S. R. et al.,2001, supra; Harms, P. A. et al., 2001, supra; Kennedy, S. et al., 2000,supra; Tomas, A. et al., 2008, supra). Therefore. in the presentinvention in order to definitively determine the pathogenicity of thechimeric PCV1-2b virus and compare it to its parental wildtype PCV2bvirus, the inventors chose the CD/CD pig model for the pathogenicitystudy. As expected, the wildtype PCV2b alone caused severe clinicalPCVAD in CD/CD pigs, resulting in the death or early euthanasia due toclinical manifestation of PCVAD in 4/10 PCV2b-infected pigs. This isconsistent with previous studies that have shown 25% mortality andsignificant clinical PCVAD in PCV2-infected CD/CD pigs (Allan, G. etal., 2003, supra; Bolin, S. R. et al., 2001, supra; Harms, P. A. et al.,2001, supra). The overall lymphoid lesion scores for PCV2b-infected pigswere consistent with moderate-to-severe systemic PCVAD (Opriessnig, T.et al. 2007, supra). The generally accepted threshold for viremia insevere PCVAD cases is 10⁷ viral genomic copies per ml of serum, thoughhost variation prevents a single definitive diagnostic cut-off(Brunborg, I. M. et al., 2004, Quantitation of porcine circovirus type 2isolated from serum/plasma and tissue samples of healthy pigs and pigswith postweaning muitisystemic wasting syndrome using a TaqMan-basedreal-time PCR, J Virol Methods 122:171-8; Olvera, A. et al., 2004,Comparison of porcine circovirus type 2 load in serum quantified by areal time PCR in postweaning muitisystemic wasting syndrome and porcinedermatitis and nephropathy syndrome naturally affected pigs, J VirolMethods 117:75-80; Segales, J. et al., 2005, Quantification of porcinecircovirus type 2 (PCV2) DNA in serum and tonsillar, nasal, tracheo

bronchial, urinary and faecal swabs of pigs with and without postweaningmuitisystemic wasting syndrome (PMWS), Vet Microbiol 111:223-9). By 21dpi, 9/10 PCV2b-infected pigs were above this threshold, compared toonly 1/10 chimeric PCV1-2b virus-infected pigs. The 4 pigs that died orwere euthanized earlier due to PCVAD all had higher PCV2 DNA viral loadsin serum and lymphoid tissues but had no detectable PCV2 IgG antibodies,indicating that these PCV2b-infected pigs died because of insufficientimmune responses and higher level of virus replication. This result isconsistent with a previously reported correlation between low antibodyresponse and increased severity of PCVAD (Meerts, P. et al., 2006,Correlation between the presence of neutralizing antibodies againstporcine circovirus 2 (PCV2) and protection against replication of thevirus and development of PCV2-associated disease, BMC Vet Res 2:6). Thedata clearly demonstrated that the PCV2b subtype used in the presentinvention is highly virulent.

The chimeric PCV1-2b virus was found to be significantly attenuated inthe CD/CD pig model, despite the fact that the CD/CD pigs wereinoculated with a dose that is at least 20-fold higher than the normalvaccination dose (Fenaux, M. et al., 2004, supra). Attenuation ofchimeric PCV1-2b virus was clearly demonstrated by all quantitative andqualitative parameters that were used to compare the chimeric PCV1-2band the wildtype PCV2b viruses. There was no mortality or morbidity seenin pigs infected with the chimeric PCV1-2b virus, compared to death andwasting seen in about half of the PCV2b-infected pigs. Microscopiclesion scores and the amounts of PCV2-specific antigen in lymphoidtissues were significantly less in the chimeric PCV1-2b-infected pigsthan in pigs infected with the wildtype PCV2b, indicating that thechimeric PCV1-2b virus causes only subclinical infection. Overall, thelymphoid lesion scores in chimeric PCV1-2b-infected pigs were notsignificantly different from the control pigs inoculated with PBSbuffer. Additionally, lower PCV2b viral load in the serum and lymphoidtissues directly correlate to the significantly less characteristiclesions or disease severity in the chimeric PCV1-2b-infected pigs, asobserved in previous studies (Brunborg, I. M. et al., 2004, supra;Dupont, K. et al., 2009, Transmission of different variants of PCV2 andviral dynamics in a research facility with pigs mingled fromPMWS-affected herds and non-affected herds, Vet Microbiol 139:219-26;Fenaux, M. et al. 2004, supra; Harding, J. C et al., 2008, Porcinecircovirus-2 DNA concentration distinguishes wasting from nonwastingpigs and is correlated with lesion distribution, severity, andnucleocapsid staining intensity, J Vet Diagn invest 20:274-82; Krakowka,S. et al., 2005, Features of porcine circovirus-2 disease: correlationsbetween lesions, amount and distribution of virus, and clinical outcome,J Vet Diagn Invest 17:213-22; Mcintosh, K. A. et al., 2009, supra;Olvera, A. et al., 2004, supra; Yang, Z. Z. et al., 2007, supra).

After demonstrating that the chimeric PCV1-2b virus is attenuated in thesusceptible and sensitive CD/CD pig model, the inventors then conducteda combined immunogenicity and challenge study in conventional SPF pigs.Three-week-old conventional cross-breed SPF pigs were chosen for theimmunogenicity/challenge experiment in order to more closely mimic fieldvaccination conditions since such a live-attenuated vaccine will beeventually used in conventional pigs. Though clinical PCVAD was notexpected in this conventional SPF model based on our earlier publishedstudies (Fenaux, M. et al., 2002, supra; Fenaux, M. et al., 2004, supra;Fenaux, M. et al., 2004, supra; Fenaux, Met al., 2003, supra, Fenaux, M.et al., 2004A, supra; Opriessnig, T, et al., 2009, Difference inseverity of porcine circovirus type two-induced pathological lesionsbetween Landrace and Pietrain pigs, J Anim Sci 87:1582-90; Opriessnig,T. et al., 2008, supra), it was anticipated that the level of viremia,viral loads and the characteristic histological lesions in lymphoidtissues induced by PCV2 in the conventional pig model are sufficientparameters for evaluating vaccine efficacy (Fenaux, M. et al., 2004,supra). As a live-attenuated vaccine, it is important to determine ifchimeric PCV1-2b virus can induce sufficient level of protectiveantibody response upon vaccination of pigs. Two of the four currentlyavailable vaccines are based on recombinant PCV2a capsid proteins, andthus PCV2 capsid-specific humoral immune response is known to beimportant for protection. The results from the present invention showedthat PCV2 capsid-specific antibodies were detected in the sera ofPCV1-2b-vaccinated pigs as early as 14 dpv, and by 28 dpv all of the 20vaccinated pigs had seroconverted to anti-PCV2 capsid-specific antibody.The antibody titers plateaued by 35 dpv and remained high at the time ofchallenge at 56 dpv, indicating that the chimeric PCV1-2b vaccine virusis capable of eliciting strong humoral immune response in conventionalpigs.

Upon challenge with either wildtype PCV2a or PCV2b subtype, conventionalSPF pigs vaccinated with the attenuated chimeric PCV1-2b virus hadsignificantly lower viral DNA loads in the serum and TBLN tissues,significantly decreased level of severity and incidence ofcharacteristic microscopic lesions, and significantly lower amounts ofPCV2-specific antigen in lymphoid tissues compared to the unvaccinatedcontrols, indicating that the chimeric PCV1-2b virus induced protectiveimmunity against wildtype virus challenge. The results also showed thatpigs vaccinated with the chimeric PCV1-2b virus were equally protectedagainst homologous challenge with PCV2b subtype and heterologouschallenge with the PCV2a subtype, as evidenced by the complete lack ofPCV2a or PCV2b viremia, significant reductions in viral 7 loads inlymphoid tissues, and significantly lower overall lymphoid lesion scoresin vaccinated pigs compared to unvaccinated controls, regardless ofchallenge virus subtype. Therefore. it appears that the newlive-attenuated chimeric PCV1-2b vaccine candidate induces bothprotective and cross-protective immunity against both PCV2 subtypes.

Some recent studies have reported that, in general, the PCV2b subtype isassociated with more severe clinical disease when compared to the PCV2asubtype (Carman, S. et al., 2008, supra; Chae, J. S, and K. S. Choi,2009, supra; Grau-Roma, L et al., 2008, A proposal on porcine circovirustype 2 (PCV2) genotype definition and their relation with postweaningmuitisystemic wasting syndrome (PMWS) occurrence, Vet Microbiol128:23-35). However, it remains debatable whether or not the PCV2bsubtype is more virulent than the PCV2a subtype, since other studiescould not definitively show a significant difference in virulencebetween PCV2a and PCV2b (An, D. J. et al., 2007, supra; Lager, K. M. etal., 2007, supra; Madson, D. M. et al., 2008, supra; Opreissnig, T. etal., 2006, supra; Opreissnig, T. et al., 2008, supra). Because of thesequence divergence between PCV2a and PCV2b, it is possible that the twosubtypes of PCV2 may differ in pathogenicity since it has been shownthat only two amino acid changes in the cap gene were sufficient toalter the pathogenicity of PCV2a (Fenaux, M. et al., 2004, Two aminoacid mutations in the capsid protein of type 2 porcine circovirus (PCV2)enhanced PCV2 replication in vitro and attenuated the virus in vivo, JViol 78:13440-6). In the unvaccinated control group in the currentstudy, where half of the pigs were challenged with PCV2a or PCV2b, theinventors did not observe any significant difference in virulencebetween groups. The challenge doses for PCV2a and PCV2b were equivalent,but there was no significant difference in PCV2 viral loads in serum andTBLN tissues, the characteristic microscopic lesion scores, or theamounts of PCV2 antigen in lymphoid tissues between the PCV2a and PCV2bchallenge groups. There were some minor differences in the antibodyresponse, including a slightly delayed seroconversion to PCV2b comparedto PCV2a in the unvaccinated pigs (FIG. 5). In the vaccinated pigs,PCV2b challenge resulted in slightly greater overall lymphoid lesionscores while the PCV2a challenge resulted in a higher number of TBLNtissues positive by qPCR. Overall, the results are consistent with otherstudies that have found no significant difference in PCV2a and PCV2bpathogenicity under experimental conditions (An, D. J. et al., 2007,supra; Lager, K. M et al., 2007, supra; Madson, D. M. et al., 2008,supra; Opriessnig, T. et al., 2006, supra; Opriessnig, T. et al., 2008,supra).

Differences in the antigenic profiles between PCV2a and PCV2b subtypeshave been reported, and it has been speculated that a lack of sufficientlevel of cross-protection by the current PCV2a-based commercial vaccinesmay have contributed to an increase in PCVAD severity associated withthe PCV2b subtype in global pig populations (Cheung, A. K. et al., 2007,sup-a; Dupont, K. et al., 2008, supra; Lekcharoensuk, P. et al., 2004,supra; Shang, S. B. et al., 2009, supra). Most of the sequencevariations between PCV2a and PCV2b appear in the capsid gene, includinga signature distinctive amino acid motif (Cheung, A. K. et al., 2007,supra; Olvera, A. et al., 2004, supra). Antibodies raised against thismotif are capable of differentiating between PCV2a and PCV2b in vitro,indicating possible differences in antigenicity (Beach et al,unpublished data). The results from the present invention showed that alive-attenuated vaccine, PCV 1-2b, based on the capsid of the new PCV2bsubtype does protect against heterologous challenge by PCV2a, thussupporting previous evidence of cross-protection of PCV2b subtypeconferred by the PCV2a-based inactivated commercial vaccines (Fort, M.,et al., 2008, supra; Fort, M. et al., 2009, supra; Opriessnig, T. etal., 2009, supra; Opriessnig, T. et al., 2008, supra).

In summary, the data from the present invention demonstrate that thechimeric PCV1-2b vaccine candidate based on the new PCV2b subtype isattenuated in CD/CD pigs and induces protective and cross-protectiveimmunity in vaccinated conventional pigs against both PCV2b and PCV2achallenge, respectively. The results from the present invention will setthe stage for further development of this chimeric PCV1-2b virus as thefirst live-attenuated vaccine against PCV2 infection and PCVAD. Althoughthe PCV1-2b vaccine virus alone offered cross-protection against bothPCV2a and PCV2b subtypes, it may be more advantageous in the future tocombine the PCV1-2b vaccine from the present invention with the currentPCV2a-based commercial vaccines for a more complete protection.

Vaccines of the infectious viral and molecular DNA clones, and methodsof using them, are also included within the scope of the presentinvention. Inoculated pigs are protected from serious viral infectionand other diseases caused by PCV2 infection or co-infection. The novelmethod protects pigs in need of protection against viral infection byadministering to the pig an immunologically effective amount of avaccine according to the invention, such as, for example, a vaccinecomprising an immunogenic amount of the infectious PCV DNA, a plasmid orviral vector containing the infectious DNA clone of PCV, the recombinantPCV DNA, the polypeptide expression products, etc. Other antigens suchas PRRSV, PPV, other infectious swine agents and immune stimulants maybe given concurrently to the pig to provide a broad spectrum ofprotection against viral infections.

The vaccines comprise, for example, the infectious viral and molecularDNA clones, the cloned PCV infectious DNA genome in suitable plasmids orvectors such as, for example, the pSCK vector, an avirulent, live virus,an inactivated virus, etc. in combination with a nontoxic,physiologically acceptable carrier and, optionally, one or moreadjuvants. The vaccine may also comprise the infectious PCV2 molecularDNA clone described herein. The infectious PCV DNA, the plasmid DNAcontaining the infectious viral genome and the live virus are preferredwith the live virus being most preferred. The avirulent, live viralvaccine of the present invention provides an advantage over traditionalviral vaccines that use either attenuated, live viruses which run therisk of reverting back to the virulent state or killed cell culturepropagated whole virus which may not induce sufficient antibody immuneresponse for protection against the viral disease.

Vaccines and methods of using them are also included within the scope ofthe present invention. Inoculated mammalian species are protected fromserious viral infection, may also provide protection for disease relatedto co-infection of PCV, such as PCVAD and porcine dermatitis andnephropathy syndrome (PDNS), and other related illness. The vaccinescomprise, for example, an inactivated or attenuated porcine TTV virus, anontoxic, physiologically acceptable carrier and, optionally, one ormore adjuvants.

The adjuvant, which may be administered in conjunction with the vaccineof the present invention, is a substance that increases theimmunological response of the pig to the vaccine. The adjuvant may beadministered at the same time and at the same site as the vaccine, or ata different time, for example, as a booster. Adjuvants also mayadvantageously be administered to the pig in a manner or at a sitedifferent from the manner or site in which the vaccine is administered.Suitable adjuvants include, but are not limited to, aluminum hydroxide(alum), immunostimulating complexes (ISCOMS), non-ionic block polymersor copolymers, cytokines (like IL-1, IL-2, IL-7, IFN-α, IFN-β, IFN-γ,etc.), saponins, monophosphoryl lipid A (MLA), muramyl dipeptides (MDP)and the like. Other suitable adjuvants include, for example, aluminumpotassium sulfate, heat-labile or heat-stable enterotoxin isolated fromEscherichia coli cholera toxin or the B subunit thereof, diphtheriatoxin, tetanus toxin, pertussis toxin, Freund's incomplete or completeadjuvant, etc. Toxin-based adjuvants, such as diphtheria toxin, tetanustoxin and pertussis toxin may be inactivated prior to use, for example,by treatment with formaldehyde.

The vaccines may further contain additional antigens to promote theimmunological activity of the infectious PCV DNA clones such as, forexample, porcine reproductive and respiratory syndrome virus (PRRSV),porcine parvovirus (PPV), other infectious swine agents and immunestimulants.

The new vaccines of this invention are not restricted to any particulartype or method of preparation. The cloned viral vaccines include, butare not limited to, infectious DNA vaccines (i.e., using plasmids,vectors or other conventional carriers to directly inject DNA intopigs), live vaccines, modified live vaccines, inactivated vaccines,subunit vaccines, attenuated vaccines, genetically engineered vaccines,etc. These vaccines are prepared by standard methods known in the art.

As a further benefit, the preferred live virus of the present inventionprovides a genetically stable vaccine that is easier to make, store anddeliver than other types of attenuated vaccines.

Another preferred vaccine of the present invention utilizes suitableplasmids for delivering the nonpathogenic DNA clone to pigs. In contrastto the traditional vaccine that uses live or killed cell culturepropagated whole virus, this invention provides for the directinoculation of pigs with the plasmid DNA containing the infectious viralgenome.

Additional genetically engineered vaccines, which are desirable in thepresent invention, are produced by techniques known in the art. Suchtechniques involve, but are not limited to, further manipulation ofrecombinant DNA, modification of or substitutions to the amino acidsequences of the recombinant proteins and the like.

Genetically engineered vaccines based on recombinant DNA technology aremade, for instance, by identifying alternative portions of the viralgene encoding proteins responsible for inducing a stronger immune orprotective response in pigs (e.g., proteins derived from ORF3, ORF4,etc.). Such identified genes or immuno-dominant fragments can be clonedinto standard protein expression vectors, such as the baculovirusvector, and used to infect appropriate host cells (see, for example,O'Reilly et al., “Baculovirus Expression Vectors: A Lab Manual,” Freeman& Co., 1992). The host cells are cultured, thus expressing the desiredvaccine proteins, which can be purified to the desired extent andformulated into a suitable vaccine product.

If the clones retain any undesirable natural abilities of causingdisease, it is also possible to pinpoint the nucleotide sequences in theviral genome responsible for any residual virulence, and geneticallyengineer the virus avirulent through, for example, site-directedmutagenesis. Site-directed mutagenesis is able to add, delete or changeone or more nucleotides (see, for instance, Zoller et al., DNA3:479-488, 1984). An oligonucleotide is synthesized containing thedesired mutation and annealed to a portion of single stranded viral DNA.The hybrid molecule, which results from that procedure, is employed totransform bacteria. Then double-stranded DNA, which is isolatedcontaining the appropriate mutation, is used to produce full-length DNAby ligation to a restriction fragment of the latter that is subsequentlytransfected into a suitable cell culture. Ligation of the genome intothe suitable vector for transfer may be accomplished through anystandard technique known to those of ordinary skill in the art.Transfection of the vector into host cells for the production of viralprogeny may be done using any of the conventional methods such ascalcium-phosphate or DEAE-dextran mediated transfection,electroporation, protoplast fusion and other well-known techniques(e.g., Sambrook et al., “Molecular Cloning: A Laboratory Manual,” ColdSpring Harbor Laboratory Press, 1989). The cloned virus then exhibitsthe desired mutation. Alternatively, two oligonucleotides can besynthesized which contain the appropriate mutation. These may beannealed to form double-stranded DNA that can be inserted in the viralDNA to produce full-length DNA.

An immunologically effective amount of the vaccines of the presentinvention is administered to a pig in need of protection against viralinfection. The immunologically effective amount or the immunogenicamount that inoculates the pig can be easily determined or readilytitrated by routine testing. An effective amount is one in which asufficient immunological response to the vaccine is attained to protectthe pig exposed to the PCV virus. Preferably, the pig is protected to anextent in which one to all of the adverse physiological symptoms oreffects of the viral disease are significantly reduced, ameliorated ortotally prevented.

The vaccine can be administered in a single dose or in repeated doses.Dosages may range, for example, from about 1 microgram to about 1,000micrograms of the plasmid DNA containing the infectious chimeric DNAgenome (dependent upon the concentration of the immuno-active componentof the vaccine), preferably 100 to 200 micrograms of the chimeric PCV1-2DNA clone, but should not contain an amount of virus-based antigensufficient to result in an adverse reaction or physiological symptoms ofviral infection. Methods are known in the art for determining ortitrating suitable dosages of active antigenic agent to find minimaleffective dosages based on the weight of the pig, concentration of theantigen and other typical factors. Preferably, the infectious viral DNAclone is used as a vaccine, or a live infectious virus can be generatedin vitro and then the live virus is used as a vaccine. In that case,from about 50 to about 10,000 of the 50% tissue culture infective dose(TCID₅₀) of live virus, for example, can be given to a pig.

The new vaccines of this invention are not restricted to any particulartype or method of preparation. The vaccines include, but are not limitedto, modified live vaccines, inactivated vaccines, subunit vaccines,attenuated vaccines, genetically engineered vaccines, etc.

The advantages of live vaccines are that all possible immune responsesare activated in the recipient of the vaccine, including systemic,local, humoral and cell-mediated immune responses. The disadvantages oflive virus vaccines, which may outweigh the advantages, lie in thepotential for contamination with live adventitious viral agents or therisk that the virus may revert to virulence in the field.

To prepare inactivated virus vaccines, for instance, the viruspropagation and virus production can occur in cultured porcine celllines such as, without limitation PK-15 cells. Serial virus inactivationis then optimized by protocols generally known to those of ordinaryskill in the art or, preferably, by the methods described herein.

Inactivated virus vaccines may be prepared by treating the procinecircovirus with inactivating agents such as formalin or hydrophobicsolvents, acids, etc., by irradiation with ultraviolet light or X-rays,by heating, etc. Inactivation is conducted in a manner understood in theart. For example, in chemical inactivation, a suitable virus sample orserum sample containing the virus is treated for a sufficient length oftime with a sufficient amount or concentration of inactivating agent ata sufficiently high (or low, depending on the inactivating agent)temperature or pH to inactivate the virus. Inactivation by heating isconducted at a temperature and for a length of time sufficient toinactivate the virus. Inactivation by irradiation is conducted using awavelength of light or other energy source for a length of timesufficient to inactivate the virus. The virus is considered inactivatedif it is unable to infect a cell susceptible to infection.

Genetically engineered vaccines, which are also desirable in the presentinvention, are produced by techniques known in the art. Such techniquesinvolve, but are not limited to, the use of RNA, recombinant DNA,recombinant proteins, live viruses and the like.

For instance, after purification, the wild-type virus may be isolatedfrom suitable clinical, biological samples such as serum, fecal, saliva,semen and tissue samples by methods known in the art, preferably by themethod taught herein using infected pigs or infected suitable celllines. The DNA is extracted from the biologically pure virus orinfectious agent by methods known in the art, and purified by methodsknown in the art, preferably by ultracentrifugation in a CsCl gradient.The cDNA of viral genome is cloned into a suitable host by methods knownin the art (see Maniatis et al., id.), and the virus genome is thenanalyzed to determine essential regions of the genome for producingantigenic portions of the virus. Thereafter. the procedure is generallythe same as that for the modified live vaccine, an inactivated vaccineor a subunit vaccine.

Genetically engineered vaccines based on recombinant DNA technology aremade, for instance, by identifying the portion of the viral gene whichencodes for proteins responsible for inducing a stronger immune orprotective response in pigs (e.g., immunogenic viral protein such ascapsid protein derived from ORF2). Such identified genes orimmuno-dominant fragments can be cloned into standard protein expressionvectors, such as the baculovirus vector, and used to infect appropriatehost cells (see, for example, O'Reilly et al., “Baculovirus ExpressionVectors: A Lab Manual,” Freeman & Co. (1992)). The host cells arecultured, thus expressing the desired vaccine proteins, which can bepurified to the desired extent and formulated into a suitable vaccineproduct.

Alternatively, DNA from the isolated porcine PCV which encode one ormore capsid proteins can be inserted into live vectors, such as apoxvirus or an adenovirus and used as a vaccine.

An immunologically effective amount of the vaccine of the presentinvention is administered to an porcine or mammalian species in need ofprotection against said infection or syndrome The “immunologicallyeffective amount” can be easily determined or readily titrated byroutine testing. An effective amount is one in which a sufficientimmunological response to the vaccine is attained to protect the pig orother mammal exposed to the porcine TTV virus which may cause PCVAD,porcine dermatitis and nephropathy syndrome (PDNS), or related illness.Preferably, the pig or other mammalian species is protected to an extentin which one to all of the adverse physiological symptoms or effects ofthe viral disease are found to be significantly reduced, ameliorated ortotally prevented.

The vaccine can be administered in a single dose or in repeated doses.Dosages may contain, for example, from 1 to 1,000 micrograms ofvirus-based antigen (dependent upon the concentration of theimmuno-active component of the vaccine), but should not contain anamount of virus-based antigen sufficient to result in an adversereaction or physiological symptoms of viral infection. Methods are knownin the art for determining or titrating suitable dosages of activeantigenic agent based on the weight of the bird or mammal, concentrationof the antigen and other typical factors.

The vaccine can be administered to pigs. Also, the vaccine can be givento humans such as pig farmers who are at high risk of being infected bythe viral agent. The vaccine can conveniently be administered orally,intrabuccally, intranasally, transdermally, parenterally, etc. Theparenteral route of administration includes, but is not limited to,intramuscular, intravenous, intraperitoneal and subcutaneous routes.

When administered as a liquid, the present vaccine may be prepared inthe form of an aqueous solution, a syrup, an elixir, a tincture and thelike. Such formulations are known in the art and are typically preparedby dissolution of the antigen and other typical additives in theappropriate carrier or solvent systems. Suitable carriers or solventsinclude, but are not limited to, water, saline, ethanol, ethyleneglycol, glycerol, etc. Typical additives are, for example, certifieddyes, flavors, sweeteners and antimicrobial preservatives such asthimerosal (sodium ethylmercurithiosalicylate). Such solutions may bestabilized, for example, by addition of partially hydrolyzed gelatin,sorbitol or cell culture medium, and may be buffered by conventionalmethods using reagents known in the art, such as sodium hydrogenphosphate, sodium di hydrogen phosphate, potassium hydrogen phosphate,potassium dihydrogen phosphate, a mixture thereof, and the like.

Liquid formulations also may include suspensions and emulsions whichcontain suspending or emulsifying agents in combination with otherstandard co-formulants. These types of liquid formulations may beprepared by conventional methods. Suspensions, for example, may beprepared using a colloid mill. Emulsions, for example, may be preparedusing a homogenizer.

Parenteral formulations, designed for injection into body fluid systems,require proper isotonicity and pH buffering to the corresponding levelsof mammalian body fluids. Isotonicity can be appropriately adjusted withsodium chloride and other salts as needed, Suitable solvents, such asethanol or propylene glycol, can be used to increase the solubility ofthe ingredients in the formulation and the stability of the liquidpreparation. Further additives which can be employed in the presentvaccine include, but are not limited to, dextrose, conventionalantioxidants and conventional chelating agents such as ethylenediaminetetraacetic acid (EDTA). Parenteral dosage forms must also be sterilizedprior to use.

TABLE 1 Oligonucleotide primers used in the construction ofinfectious DNA clones of PCV2b and chimeric PCV1-2b Amplicon PrimerSequence (5′→3′) size (bp) A TTT CCG CGG GCT GGC TGA ACT TTT GAA AG1,779 B AGC CCG CGG AAA TTT CTG ACA AAC GTT AC CCGT AAT GGT TTT TAT TTT TAA GGG TTA AGT GG   718 DCTT TCA CTT TTA TAG GAT GAC GTA TCC AAG GAG G ETTC GGG TAC CCG AAG GCC GAT T   122 FCAC TTA ACC CTT AAA AAT AAA AAC CAT TAC GAT GCCT CCT TGG ATA CGT CAT CCT ATA AAA GTG AAA G 1,043 HCAG TGG ATC CCC CGG GCT GCA GGA

TABLE 2 Distribution of histopathological lesions in tissues ofcaesarean-derived colostrum-deprived (CD/CD) pigs experimentallyinoculated with PBS buffer, PCV1-2b chimeric virus, and wildtype PCV2bvirus No. of pigs with lesions/no. examined (mean score) Lymph nodesSpleen Tonsil dpi Group Inoculum Lung Liver Thymus Heart Kidney IleumColon LD^(b) HR^(c) LD^(b) HR^(c) LD^(b) HR^(c) 21/nec^(a) 1 PBS 3/5 1/50/5 0/5 0/5 0/5 0/5 1/5 0/5^(I) 0/5 0/5 0/5^(I) 0/5^(I) (0.8) (0.4)(0.2)^(I) 2 PCV1-2b 4/5 3/5 1/5 0/5 2/5 1/5 0/5 2/5 1/5 1/5 1/5 1/5 1/5(1.0) (1.0) (0.2) (0.6) (0.2) (0.6)^(I,II) (0.4)^(I,II) (0.2) (0.2)(0.2)^(I,II) (0.4)^(I,II) 3 PCV2b 5/5 5/5 2/5 1/5 3/5 4/5 3/5 5/5 5/54/5 4/5 5/5 5/5 (2.8) (2.4) (0.6) (0.2) (0.8) (2.0) (1.0) (2.6)^(II)(2.2)^(II) (2.0) (1.8) (2.4)^(II) (2.2)^(II) 42/nec^(a) 1 PBS 0/50/5^(I) 0/5 0/5 0/5^(I) 0/5 0/5 0/5^(I) 0/5 0/5 0/5 0/5 0/5 2 PCV1-2b4/5 4/5 0/5 3/5 3/5 1/5 0/5 2/5 1/5 0/5 0/5 1/5 1/5 (1.0) (0.8)^(I,II)(0.6) (1.6)^(I,II) (0.2) (0.4)^(I,II) (0.4) (0.2) (0.2) 3 PCV2b 3/5 5/52/4^(d) 1/5 5/5 4/5 4/5 5/5 4/5 3/5 3/5 4/5 3/5 (2.0) (1.4)^(II) (0.75)(0.2) (2.6)^(II) (1.6) (1.0) (2.2)^(II) (2.0) (1.2) (1.0) (2.0) (1.8)^(a)pigs that died or were euthanized early due to PCVAD were includedin the analysis ^(b)lymphoid depletion ^(c)histiocytic replacement^(d)thymus was not present in one of the pigs tested ^(I, II)groups thathave statistically significant differences in group median score havedifferent numerals

TABLE 3 Immunohistochemical detection of PCV2 capsid-specific antigen incaesarean-derived colostrum-deprived (CD/CD) experimentally inoculatedwith PBS buffer, PCV1-2b chimeric virus, and PCV2b wildtype virus No, ofpigs positive/no. tested (mean score) dpi Group Inoculum Lung LiverThymus Heart Kidney Ileum Colon Lymph nodes Spleen Tonsil 21/nec^(a) 1PBS 0/5^(I) 0/5^(I) 0/5 0/5 0/5 0/5 0/5 0/5^(I) 0/5 0/5^(I) 2 PCV1-2b0/5^(I) 1/5 (0.2)^(I,II) 1/5 0/5 1/5 1/5 1/5 1/5 1/5 4/5 (0.2) (0.2)(0.2) (0.2) (0.4)^(I,II) (0.2) (1.0)^(I,II) 3 PCV2b 5/5 5/5 4/5 1/5 2/54/5 3/5 5/5 4/5 5/5 (1.6)^(II) (1.4)^(II) (1.6) (0.2) (0.4) (2.0) (1.6)(2.2)^(II) (1.6) (2.4)^(II) 42/nec^(a) 1 PBS 0/5 0/5 0/5 0/5 0/5 0/5 1/50/5^(I) 0/5 0/5^(I) (0.2) 2 PCV1-2b 0/5 0/5 0/5 0/5 1/5 0/5 0/5 4/5 1/51/5 (0.2) (0.8)^(I,II) (0.2) (0.2)^(I,II) 3 PCV2b 3/5 3/5 2/4^(b) 1/54/5 4/5 3/5 5/5 3/5 5/5 (1.6) (1.4) (1.5) (0.2) (1.8) (1.8) (1.2)(2.2)^(II) (1.4) (2.2)^(II) ^(a)Pigs that died or were euthanized earlydue to PCVAD were included in the analysis ^(b)thymus was not present inone of the pigs tested ^(I,II)groups that have statistically significantdifferences in group median score have different numerals

TABLE 4 Microscopic lesions and PCV2-specific antigen in lymphoidtissues during necropsy in conventional pigs vaccinated with PBS bufferor chimeric PCV1-2b candidate vaccine and subsequently challenged withPCV2a or PCV2b wildtype viruses no. of pigs with lesions/no. examined(mean score) Inoculum Challenge Lymph nodes Spleen Tonsil Group (0 dpv)(56 dpv) LD^(a) HR^(b) IHC^(c) LD^(a) HR^(b) IHC^(c) LD^(a) HR^(b)IHC^(c) 1 PCV1-2b PCV2b 8/10 3/10 2/10 7/10 5/10 1/10 2/10 0/10 0/10(0.9)^(I) (0.3)^(I) (0.2) (0.7) (0.5) (0.1) (0.2) 2 PBS PCV2b 9/9 9/96/9 5/9 2/9 0/9 0/9 0/9 1/9 (1.7)^(II) (1.6)^(II) (0.8) (0.6) (0.3)(0.1) 3 PCV1-2b PCV2a-40895 0/10^(I) 0/10 0/10^(I) 0/10^(I) 0/10 0/100/10 1/10 0/10 (0.1) 4 PBS PCV2a-40895 9/10 3/10 7/10 6/10 0/10 0/102/10 0/10 4/10 (1.1)^(II) (0.4) (1.1)^(II) (0.6)^(II) (0.2) (0.4)^(a)lymphoid depletion ^(b)histiocytic replacement ^(c)detection ofPCV2-specific antigen by immunohistochernisthy (IHC); no. of pigs withpositive IHC/no. examined (mean IHC antigen score) ^(I,II)pairs oftreatments that have statistically significent differences in groupmedian score have different numerals

1-27. (canceled)
 28. A method of immunizing a pig against PCV2 viralinfection, comprising administering to the pig an immunologicallyeffective amount of a viral vaccine comprising a physiologicallyacceptable carrier and an immunogenic amount of a member selected fromthe group consisting of: (a) a chimeric PCV which contains a nucleicacid molecule of PCV comprising a nucleic acid molecule encoding achimeric, nonpathogenic PCV derived from a genomic sequence of PCV 1,and at least a portion of an encoding sequence of a capsid protein of aPCV2b strain; and (b) an inactivated chimeric PCV, comprising at least aportion of a capsid protein of a PCV2b strain.
 29. The method accordingto claim 28, comprising administering live attenuated chimeric PCV virusto the pig.
 30. The method according to claim 28, comprisingadministering the inactivated chimeric PCV virus to the pig.
 31. Themethod according to claim 28, comprising administering the vaccineparenterally, intranasally, intradermally, or transdermally to the pig.32. The method according to claim 28, comprising administering thevaccine intralymphoidly or intramuscularly to the pig.
 33. The methodaccording to claim 28, wherein an adjuvant is administered inconjunction with the viral vaccine.
 34. The method according to claim28, wherein the administration is in a single dose or in repeated doses.35. A method of protecting a pig against porcine circovirus-associateddisease (PCVAD), comprising administering to the pig an immunologicallyeffective amount of a viral vaccine comprising a physiologicallyacceptable carrier and an immunogenic amount of a member selected fromthe group consisting of: (a) a chimeric PCV which contains a nucleicacid molecule of PCV comprising a nucleic acid molecule encoding achimeric, nonpathogenic PCV derived from a genomic sequence of PCV1, andat least a portion of an encoding sequence of a capsid protein of aPCV2b strain; and (b) an inactivated chimeric PCV, comprising at least aportion of a capsid protein of a PCV2b strain.
 36. The method accordingto claim 35, comprising administering live attenuated chimeric PCV virusto the pig.
 37. The method according to claim 35, comprisingadministering the inactivated chimeric PCV virus to the pig.
 38. Themethod according to claim 35, comprising administering the vaccineparenterally, intranasally, intradermally, or transdermally to the pig.39. The method according to claim 35, comprising administering thevaccine intralymphoidly or intramuscularly to the pig.
 40. The methodaccording to claim 35, wherein an adjuvant is administered inconjunction with the viral vaccine.
 41. The method according to claim35, wherein the administration is in a single dose or in repeated doses.